Polymer grafting by polysaccharide synthases

ABSTRACT

The present invention relates to methodology for polymer grafting by a polysaccharide synthase and, more particularly, polymer grafting using the hyaluronate synthase from  Pasteurella multocida . The present invention also relates to coatings for biomaterials wherein the coatings provide protective properties to the biomaterial and/or act as a bioadhesive. Such coatings could be applied to electrical devices, sensors, catheters and any device which may be contemplated for use within a mammal. The present invention further relates to drug delivery matrices which are biocompatible and may comprise combinations of a biomaterial or a bioadhesive and a medicament or a medicament-containing liposome. The biomaterial and/or bioadhesive is a hyaluronic acid polymer produced by a hyaluronate synthase from  Pasteurella multocida . The present invention also relates to the creation of chimeric molecules containing hyaluronic acid or hyaluronic acid—like chains attached to various compounds and especially carbohydrates or hydroxyl containing substances. The present invention also relates to a chondroitan synthase from  Pasteurella multocida  which is capable of producing polysaccharide polymers on an acceptor or primer molecule.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation of co-pending U.S. patentapplication Ser. No. 09/437,277, entitled “POLYMER GRAFTING BYPOLYSACCHARIDE SYNTHASES,” filed Nov. 10, 1999; which claims benefitunder 35 U.S.C. 119(e) of U.S. Provisional Application Serial No.60/107,929, filed Nov. 11, 1998; and is a continuation-in-part of U.S.patent application Ser. No. 09/283,402, entitled “DNA ENCODINGHYALURONAN SYNTHASE FROM PASTEURELLA MULTOCIDA AND METHODS,” filed Apr.1, 1999, all of which are hereby expressly incorporated herein in theirentirety by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] Not applicable.

BACKGROUND

[0003] 1. Field of the Invention

[0004] The present invention relates to methodology for polymer graftingby a polysaccharide synthase and, more particularly, polymer graftingusing the hyaluronate synthase from Pasteurella multocida. The presentinvention also relates to coatings for biomaterials wherein the coatingsprovide protective properties to the biomaterial and/or act as abioadhesive. Such coatings could be applied to electrical devices,sensors, catheters and any device which may be contemplated for usewithin a mammal. The present invention further relates to drug deliverymatrices which are biocompatible and may comprise combinations of abiomaterial or a bioadhesive and a medicament or a medicament-containingliposome. The biomaterial and/or bioadhesive is a hyaluronic acidpolymer produced by a hyaluronate synthase from Pasteurella multocida.The present invention also relates to the creation of chimeric moleculescontaining hyaluronic acid or hyaluronic acid—like chains orglycosaminoglycan chains attached to various compounds and especiallycarbohydrates or hydroxyl containing substances.

[0005] 2. Description of the Related Art

[0006] Polysaccharides are large carbohydrate molecules composed fromabout 25 sugar units to thousands of sugar units. Animals, plants, fungiand bacteria produce an enormous variety of polysaccharide structureswhich are involved in numerous important biological functions such asstructural elements, energy storage, and cellular interaction mediation.Often, the polysaccharide's biological function is due to theinteraction of the polysaccharide with proteins such as receptors andgrowth factors. The glycosaminoglycan class of polysaccharides, whichincludes heparin, chondroitan, and hyaluronic acid, play major roles indetermining cellular behavior (e.g. migration, adhesion) as well as therate of cell proliferation in mammals. These polysaccharides are,therefore, essential for correct formation and maintenance of organs ofthe human body.

[0007] Several species of pathogenic bacteria and fungi also takeadvantage of the polysaccharide's role in cellular communication. Thesepathogenic microbes form polysaccharide surface coatings or capsulesthat are identical or chemically similar to host molecules. Forinstance, Group A & C Streptococcus and Type A Pasteurella multocidaproduce authentic hyaluronic acid capsules and pathogenic Escherichiacoli are known to make capsules composed of polymers very similar tochondroitan and heparin. The pathogenic microbes form the polysaccharidesurface coatings or capsules because such a coating is nonimmunogenicand protects the bacteria from host defenses thereby providing theequivalent of molecular camouflage.

[0008] Enzymes alternatively called synthases, synthetases, ortransferases, catalyze the polymerization of polysaccharides found inliving organisms. Many of the known enzymes also polymerize activatedsugar nucleotides. The most prevalent sugar donors contain UDP but ADP,GDP, and CMP are also used depending on (1) the particular sugar to betransferred and (2) the organism. Many types of polysaccharides arefound at, or outside of, the cell surface. Accordingly, most of thesynthase activity is typically associated with either the plasmamembrane on the cell periphery or the Golgi apparatus membranes that areinvolved in secretion. In general, these membrane-bound synthaseproteins are difficult to manipulate by typical procedures and only afew enzymes have been identified after biochemical purification.

[0009] A larger number of synthases have been cloned and sequenced atthe nucleotide level using ‘reverse genetic’ approaches in which thegene or the complimentary DNA (cDNA) was obtained before the protein wascharacterized. Despite this sequence information, the molecular detailsconcerning the three-dimensional native structures, the active sites,and the mechanisms of catalytic action of the polysaccharide synthases,in general, are very limited or absent. For example, the catalyticmechanism for glycogen synthesis is not yet known in detail even thoughthe enzyme was discovered decades ago. In another example, it is still amatter of debate whether the enzymes that produce heteropolysaccharidesutilize one UDP-sugar binding site to transfer both precursors, oralternatively, if there exists two dedicated regions for each substrate.

[0010] A wide variety of polysaccharides are commercially harvested frommany sources, such as xanthan from bacteria, carrageenans from seaweed,and gums from trees. This substantial industry supplies thousands oftons of these raw materials for a multitude of consumer products rangingfrom ice cream desserts to skin cream cosmetics. Vertebrate tissues andpathogenic bacteria are the sources of more exotic polysaccharidesutilized in the medical field as surgical aids, vaccines, andanticoagulants. For example, two glycosaminoglycan polysaccharides,heparin from pig intestinal mucosa and hyaluronic acid from roostercombs, are employed in several applications including clot preventionand eye surgery, respectively. Polysaccharides extracted from bacterialcapsules (e.g. various Streptococcus pneumoniae strains) are utilized tovaccinate both children and adults against disease with varying levelsof success. However, for the most part, one must use the existingstructures found in the raw materials as obtained from nature. In manyof the older industrial processes, chemical modification (e.g.hydrolysis, sulfation, deacetylation) is used to alter the structure andproperties of the native polysaccharide. However, the synthetic controland the reproducibility of large-scale reactions are not alwayssuccessful.

[0011] Some of the current methods for designing and constructingcarbohydrate polymers in vitro utilize: (i) difficult, multistep sugarchemistry, or (ii) reactions driven by transferase enzymes involved inbiosynthesis, or (iii) reactions harnessing carbohydrate degradingenzymes catalyzing transglycosylation. The latter two methods arerestricted by the specificity and the properties of the availablenaturally occurring enzymes. Many of these enzymes are neitherparticularly abundant nor stable but are almost always expensive.Overall, the procedures currently employed yield polymers containingbetween 2 and about 12 sugars. Unfortunately, many of the physical andbiological properties of polysaccharides do not become apparent untilthe polymer contains 25, 100, or even thousands of monomers.

[0012] As stated above, polysaccharides are the most abundantbiomaterials on earth, yet many of the molecular details of theirbiosynthesis and function are not clear. Hyaluronic acid or “HA” is alinear polysaccharide of the glycosaminoglycan class and is composed ofup to thousands of β(1,4)GlcUA-β(1,3)GlcNAc repeats. In vertebrates, HAis a major structural element of the extracellular matrix and playsroles in adhesion and recognition. HA has a high negative charge densityand numerous hydroxyl groups, therefore, the molecule assumes anextended and hydrated conformation in solution. The viscoelasticproperties of cartilage and synovial fluid are, in part, the result ofthe physical properties of the HA polysaccharide. HA also interacts withproteins such as CD44, RHAMM, and fibrinogen thereby influencing manynatural processes such as angiogenesis, cancer, cell motility, woundhealing, and cell adhesion.

[0013] There are numerous medical applications of HA. For example, HAhas been widely used as a viscoelastic replacement for the vitreoushumor of the eye in ophthalmic surgery during implantation ofintraocular lenses in cataract patients. HA injection directly intojoints is also used to alleviate pain associated with arthritis.Chemically cross-linked gels and films are also utilized to preventdeleterious adhesions after abdominal surgery. Other researchers usingother methods have demonstrated that adsorbed HA coatings also improvethe biocompatibility of medical devices such as catheters and sensors byreducing fouling and tissue abrasion.

[0014] HA is also made by certain microbes that cause disease in humansand animals. Some bacterial pathogens, namely Gram-negative Pasteurellamultocida Type A and Gram-positive Streptococcus Group A and C, producean extracellular HA capsule which protects the microbes from hostdefenses such as phagocytosis. Mutant bacteria that do not produce HAcapsules are 10²- and 10³-fold less virulent in comparison to theencapsulated strains. Furthermore, the Paramecium bursaria chlorellavirus (PBCV-1) directs the algal host cells to produce a HA surfacecoating early in infection.

[0015] The various HA synthases (“HAS”), the enzymes that polymerize HA,utilize UDP-GlcUA and UDP-GlcNAc sugar nucleotide precursors in thepresence of a divalent Mn or Mg ion to polymerize long chains of HA. TheHA chains can be quite large (n=10² to 10⁴). In particular, the HASs aremembrane proteins localized to the lipid bilayer at the cell surface.During HA biosynthesis, the HA polymer is transported across the bilayerinto the extracellular space. In all HASs, a single species ofpolypeptide catalyzes the transfer of two distinct sugars. In contrast,the vast majority of other known glycosyltransferases transfer only onemonosaccharide.

[0016] HasA (or SPHAS) from Group A Streptococcus pyogenes was the firstHA synthase to be described at the molecular level. The variousvertebrate homologs (Xenopus frog DG42 or XIHAS1; murine and human HAS1, HAS2, and HAS3) and the viral enzyme, A98R, are quite similar at theamino acid level to certain regions of the HasA polypeptide chain (˜30%identity overall). At least 7 short motifs (5-9 residues) interspersedthroughout these enzymes are identical or quite conserved. Theevolutionary relationship among these HA syntheses from such dissimilarsources is not clear at present. The enzymes are predicted to have asimilar overall topology in the bilayer: membrane-associated regions atthe amino and the carboxyl termini flank a large cytoplasmic centraldomain (˜200 amino acids). The amino terminal region appears to containtwo transmembrane segments while the carboxyl terminal region appears tocontain three to five membrane-associated or transmembrane segmentsdepending on the species. Very little of these HAS polypeptide chainsare expected to be exposed to the outside of the cell.

[0017] With respect to the reaction pathway utilized by this group ofenzymes, mixed findings have been reported from indirect experiments.The Group A streptococcal enzyme was reported to add sugars to thenonreducing terminus of the growing chain as determined by selectivelabeling and degradation studies. Using a similar approach, however, twolaboratories working with the enzyme preparations from mammalian cellsconcluded that the new sugars were added to the reducing end of thenascent chain. In comparing these various studies, the analysis of theenzymatically-released sugars from the streptococcal system added morerigorous support for their interpretation. In another type ofexperiment, HA made in mammalian cells was reported to have a covalentlyattached UDP group as measured by an incorporation of low amounts ofradioactivity derived from ³²P-labeled UDP-sugar into an anionicpolymer. This data implied that the last sugar was transferred to thereducing end of the polymer. Thus, it remains unclear if these rathersimilar HAS polypeptides from vertebrates and streptococci actuallyutilize different reaction pathways.

[0018] To facilitate the development of biotechnological medicalimprovements, the present invention provides a method to apply a surfacecoating of HA that will shield the artificial components or compoundsfrom the detrimental responses of the body as well as encourageengrafting of a foreign medical device within living tissue. Such acoating of HA will bridge the gap between man-made substances and livingflesh (i.e. improve biocompatibilty). The HA can also be used as abiomaterial such as a biodhesive or a bioadhesive containing amedicament delivery system, such as a liposome, and which isnon-immunogenic. The present invention also encompasses the methodologyof polysaccharide polymer grafting, i.e. HA or chondroitan, using eithera hyaluronate synthase (PmHAS) or a chondroitan synthase (PmCS) from P.multocida. Modified versions of the PmHAS or PmCS enzymes (genetic orchemical) can also be utilized to graft on polysaccharides of varioussize and composition.

SUMMARY OF THE INVENTION

[0019] A unique HA synthase, PmHAS, from the fowl cholera pathogen, TypeA P. multocida has been identified and cloned and is disclosed andclaimed in co-pending U.S. Ser. No. 09/283,402, filed Apr. 1, 1999, andentitled “DNA Encoding Hyaluronan Synthase From Pasteurella Multocidaand Methods,” the contents of which are hereby expressly incorporatedherein. Expression of this single 972-residue protein allows Escherichiacoli host cells to produce HA capsules in vivo; normally E. coli doesnot make HA. Extracts of recombinant E. coli., when supplied with theappropriate UDP-sugars, make HA in vitro. Thus, the PmHAS is anauthentic HA synthase.

[0020] It has also been determined that the PmHAS adds sugars to thenonreducing end of a growing polymer chain. The correct monosaccharidesare added sequentially in a stepwise fashion to the nascent chain or asuitable exogenous HA oligosaccharide. The PmHAS sequence, however, issignificantly different from the other known HA syntheses. There appearsto be only two short potential sequence motifs ([D/N]DGS[S/T];DSD[D/T]Y) in common between PmHAS and the Group A HAS—HasA. Instead, aportion of the central region of the new enzyme is more homologous tothe amino termini of other bacterial glycosyltransferases that producedifferent capsular polysaccharides or lipopolysaccharides. Furthermore,even though PmHAS is about twice as long as any other HAS enzyme, itonly has two predicted transmembrane spanning helices separated by ˜320residues. Thus at least a third of the polypeptide is predicted not tobe in the cytoplasm.

[0021] When the PmHAS is given long elongation reaction times, HApolymers of at least 400 sugars long are formed. Unlike any other knownHAS enzyme, PmHAS also has the ability to extend exogenously suppliedshort HA oligosaccharides into long HA polymers in vitro. If enzyme issupplied with these short HA oligosaccharides, total HA biosynthesis isincreased up to 50-fold over reactions without the exogenousoligosaccharide. The nature of the polymer retention mechanism of thePmHAS polypeptide might be the causative factor for this activity: i.e.a HA-binding site may exist that holds onto the HA chain duringpolymerization. Small HA oligosaccharides also, are capable of occupyingthis site of the recombinant enzyme and thereafter be extended intolonger polysaccharide chains.

[0022] Most membrane proteins are relatively difficult to study due totheir insolubility in aqueous solution, and the HASs are no exception.Only the enzyme from Group A and C Streptococcus bacteria has beendetergent-solubilized and purified in an active state in smallquantities. Once isolated in a relatively pure state, the streptococcalenzyme has very limited stability. A soluble recombinant form of theenzyme from P. multocida called PmHAS-D which comprises residues 1-703of the 972 residues of the native PmHAS enzyme, the amino acid sequenceof PmHAS-D is shown in SEQ ID NO:1 with the nucleotide sequence ofPmHAS-D is shown in SEQ ID NO: 2. PmHAS-D can be mass-produced in E.coli and purified by chromatography. The PmHAS-D enzyme retains theability of the parent enzyme to add on a long HA polymer onto short HAprimers. Furthermore, the purified PmHAS-D enzyme is stable in anoptimized buffer for days on ice and for hours at normal reactiontemperatures. One formulation of the optimal buffer consists of 1Methylene glycol, 0.1-0.2 M ammonium sulfate, 50 mM Tris, pH 7.2, andprotease inhibitors which allows the stability and specificity attypical reaction conditions for sugar transfer. For the reactionUDP-sugars and manganese (10-20 mM) are added. PmHAS-D will also add ona HA polymer onto plastic beads with an immobilized short HA primer.

[0023] The present invention encompasses methods of producing a varietyof unique biocompatible molecules and coatings based on polysaccharides.Polysaccharides, especially those of the glycosaminoglycan class, servenumerous roles in the body as structural elements and signalingmolecules. By grafting or making hybrid molecules composed of more thanone polymer backbone, it is possible to meld distinct physical andbiological properties into a single molecule without resorting tounnatural chemical reactions or residues.

[0024] The present invention also incorporates the propensity of certainrecombinant enzymes, when prepared in a virgin state, to utilize variousacceptor molecules as the seed for further polymer growth: naturallyoccurring forms of the enzyme or existing living host organisms do notdisplay this ability. Thus, the present invention results in (a) theproduction of hybrid polysaccharides and (b) the formation ofpolysaccharide coatings. Such hybrid polymers can serve as “molecularglue”—i.e. when two cell types or other biomaterials interact with eachhalf of a hybrid molecule, then each of the two phases are bridged.

[0025] Such polysaccharide coatings are useful for integrating a foreignobject within a surrounding tissue matrix. For example, a prostheticdevice is more firmly attached to the body when the device is coatedwith a naturally adhesive polysaccharide. Additionally, the devicesartificial components could be masked by the biocompatible coating toreduce immunoreactivity or inflammation. Another aspect of the presentinvention is the coating or grafting of HA onto various drug deliverymatrices or bioadhesives or suitable medicaments to improve and/or alterdelivery, half-life, persistence, targeting and/or toxicity.

DESCRIPTION OF THE DRAWINGS

[0026]FIG. 1 is a graphical representation showing that an HA tetramerstimulates PmHAS polymerization.

[0027]FIG. 2 is a graphical plot showing that HA polymerization iseffected by HA oligosaccharides.

[0028]FIG. 3 is a graphical plot showing HA tetramer elongation intolarger polymers by PmHAS-D.

[0029] FIGS. 4 is a graphical representation of a thin layerchromatography analysis of PmHAS extension of HA tetramer.

[0030]FIG. 5 is a graphical representation of thin layer chromatographyanalysis of the early stages of HA elongation.

[0031]FIG. 6 is an electrophoresis gel showing the purification ofPmHAS-D.

[0032]FIG. 7 is a pictorial representation of the PmHAS-D mutants.

[0033]FIG. 8 is a graphical representation of a mutant combinationassay.

[0034]FIG. 9 is a tabular representation showing enzyme activity of thePmHAS-D mutants.

[0035]FIG. 10 is a schematic representation of first generation of HAcoating on silicon.

[0036]FIG. 11 is a graphical representation of a high-throughput assayfor PmHAS-D mutants.

DETAILED DESCRIPTION OF THE INVENTION

[0037] Before explaining at least one embodiment of the invention indetail, it is to be understood that the invention is not limited in itsapplication to the details of construction and the arrangements of thecomponents set forth in the following description or illustrated in thedrawings. The invention is capable of other embodiments or of beingpracticed or carried out in various ways. Also, it is to be understoodthat the phraseology and terminology employed herein is for purpose ofdescription and should not be regarded as limiting.

[0038] As used herein, the term “nucleic acid segment” and “DNA segment”are used interchangeably and refer to a DNA molecule which has beenisolated free of total genomic DNA of a particular species. Therefore, a“purified” DNA or nucleic acid segment as used herein, refers to a DNAsegment which contains a Hyaluronate Synthase (“HAS”) coding sequence orChondroitin Synthase (“CS”) coding sequence yet is isolated away from,or purified free from, unrelated genomic DNA, for example, totalPasteurella multocida. Included within the term “DNA segment”, are DNAsegments and smaller fragments of such segments, and also recombinantvectors, including, for example, plasmids, cosmids, phage, viruses, andthe like.

[0039] Similarly, a DNA segment comprising an isolated or purifiedPmHAS-D or PmCS gene refers to a DNA segment including HAS orchondroitin synthase coding sequences isolated substantially away fromother naturally occurring genes or protein encoding sequences. In thisrespect, the term “gene” is used for simplicity to refer to a functionalprotein, polypeptide or peptide encoding unit. As will be understood bythose in the art, this functional term includes genomic sequences, cDNAsequences or combinations thereof. “Isolated substantially away fromother coding sequences” means that the gene of interest, in this casePmHAS-D or PmCS, forms the significant part of the coding region of theDNA segment, and that the DNA segment does not contain large portions ofnaturally-occurring coding DNA, such as large chromosomal fragments orother functional genes or DNA coding regions. Of course, this refers tothe DNA segment as originally isolated, and does not exclude genes orcoding regions later added to, or intentionally left in the segment bythe hand of man.

[0040] Due to certain advantages associated with the use of prokaryoticsources, one will likely realize the most advantages upon isolation ofthe HAS or chondroitin synthase gene from the prokaryote P. multocida.One such advantage is that, typically, eukaryotic enzymes may requiresignificant post-translational modifications that can only be achievedin a eukaryotic host. This will tend to limit the applicability of anyeukaryotic HAS or chondroitin synthase gene that is obtained. Moreover,those of ordinary skill in the art will likely realize additionaladvantages in terms of time and ease of genetic manipulation where aprokaryotic enzyme gene is sought to be employed. These additionaladvantages include (a) the ease of isolation of a prokaryotic genebecause of the relatively small size of the genome and, therefore, thereduced amount of screening of the corresponding genomic library and (b)the ease of manipulation because the overall size of the coding regionof a prokaryotic gene is significantly smaller due to the absence ofintrons. Furthermore, if the product of the PmHAS-D or PmCS gene (i.e.,the enzyme) requires posttranslational modifications, these would bestbe achieved in a similar prokaryotic cellular environment (host) fromwhich the gene was derived.

[0041] Preferably, DNA sequences in accordance with the presentinvention will further include genetic control regions which allow theexpression of the sequence in a selected recombinant host. Of course,the nature of the control region employed will generally vary dependingon the particular use (e.g., cloning host) envisioned.

[0042] In particular embodiments, the invention concerns isolated DNAsegments and recombinant vectors incorporating DNA sequences whichencode a PmHAS-D or PmCS gene, that includes within its amino acidsequence an amino acid sequence in accordance with SEQ ID NO:1 or 3,respectively. Moreover, in other particular embodiments, the inventionconcerns isolated DNA segments and recombinant vectors incorporating DNAsequences which encode a gene that includes within its amino acidsequence the amino acid sequence of an HAS or chondroitin synthase geneor DNA, and in particular to an HAS or chondroitin synthase gene orcDNA, corresponding to Pasteurella multocida HAS or chondroitinsynthase. For example, where the DNA segment or vector encodes a fulllength HAS or chondroitin synthase protein, or is intended for use inexpressing the HAS or chondroitin synthase protein, preferred sequencesare those which are essentially as set forth in SEQ ID NO:1 or 3,respectively.

[0043] Truncated PmHAS-D also falls within the definition of preferredsequences as set forth in SEQ ID NO:1. For instance, at the carboxylterminus, approximately 270-272 amino acids may be removed from thesequence and still have a functioning HAS. Those of ordinary skill inthe art would appreciate that simple amino acid removal from either endof the PmHAS-D sequence can be accomplished. The truncated versions ofthe sequence simply have to be checked for HAS activity in order todetermine if such a truncated sequence is still capable of producingHAS.

[0044] Nucleic acid segments having HAS or chondroitin synthase activitymay be isolated by the methods described herein. The term “a sequenceessentially as set forth in SEQ ID NO:X means that the sequencesubstantially corresponds to a portion of SEQ ID NO:X and has relativelyfew amino acids which are not identical to, or a biologically functionalequivalent of, the amino acids of SEQ ID NO:X. The term “biologicallyfunctional equivalent” is well understood in the art and is furtherdefined in detail herein, as a gene having a sequence essentially as setforth in SEQ ID NO:X, and that is associated with the ability ofprokaryotes to produce HA or a hyaluronic acid coat or chondroitin. Inthe above examples “X” refers to either SEQ ID NO: 1, 2, 3, or 4.

[0045] The art is replete with examples of practitioners ability to makestructural changes to a nucleic acid segment (i.e. encoding conserved orsemi-conserved amino acid substitutions) and still preserve itsenzymatic or functional activity. See for example: (1) Risler et al.“Amino Acid Substitutions in Structurally Related Proteins. A PatternRecognition Approach.” J. Mol. Biol. 204:1019-1029 (1988) [“ . . .according to the observed exchangeability of amino acid side chains,only four groups could be delineated; (i) Ile and Val; (ii) Leu and Met,(iii) Lys, Arg, and Gin, and (iv) Tyr and Phe.”]; (2) Niefind et al.“Amino Acid Similarity Coefficients for Protein Modeling and SequenceAlignment Derived from Main-Chain Folding Anoles.”]. Mol. Biol.219:481-497 (1991) [similarity parameters allow amino acid substitutionsto be designed]; and (3) Overington et al. “Environment-Specific AminoAcid Substitution Tables: Tertiary Templates and Prediction of ProteinFolds,” Protein Science 1:216-226 (1992) [“Analysis of the pattern ofobserved substitutions as a function of local environment shows thatthere are distinct patterns . . . ” Compatible changes can be made.]

[0046] These references and countless others, indicate that one ofordinary skill in the art, given a nucleic acid sequence, could makesubstitutions and changes to the nucleic acid sequence without changingits functionality. Also, a substituted nucleic acid segment may behighly identical and retain its enzymatic activity with regard to itsunadulterated parent, and yet still fail to hybridize thereto.

[0047] The invention discloses nucleic acid segments encoding anenzymatically active HAS or chondroitin synthase from P. multocida—PmHASand PmCS, respectively. One of ordinary skill in the art wouldappreciate that substitutions can be made to the PmHAS or PmCS nucleicacid segment listed in SEQ ID NO:2 and 4, respectively, withoutdeviating outside the scope and claims of the present invention.Standardized and accepted functionally equivalent amino acidsubstitutions are presented in Table I. TABLE I Conservative and Semi-Amino Acid Group Conservative Substitutions NonPolar R Groups Alanine,Valine, Leucine, Isoleucine, Proline, Methionine, Phenylalanine,Tryptophan Polar, but uncharged, R Groups Glycine, Serine, Threonine,Cysteine, Asparagine, Glutamine Negatively Charged R Groups AsparticAcid, Glutamic Acid Positively Charged R Groups Lysine, Arginine,Histidine

[0048] Another preferred embodiment of the present invention is apurified nucleic acid segment that encodes a protein in accordance withSEQ ID NO:1 or 3, respectively, further defined as a recombinant vector.As used herein, the term “recombinant vector” refers to a vector thathas been modified to contain a nucleic acid segment that encodes an HASor chondroitin synthase protein, or fragment thereof. The recombinantvector may be further defined as an expression vector comprising apromoter operatively linked to said HAS encoding nucleic acid segment.

[0049] A further preferred embodiment of the present invention is a hostcell, made recombinant with a recombinant vector comprising an HAS orchondroitin synthase gene. The preferred recombinant host cell may be aprokaryotic cell. In another embodiment, the recombinant host cell is aeukaryotic cell. As used herein, the term “engineered” or “recombinant”cell is intended to refer to a cell into which a recombinant gene, suchas a gene encoding HAS or chondroitin synthase, has been introduced.Therefore, engineered cells are distinguishable from naturally occurringcells which do not contain a recombinantly introduced gene. Engineeredcells are thus cells having a gene or genes introduced through the handof man. Recombinantly introduced genes will either be in the form of acDNA gene, a copy of a genomic gene, or will include genes positionedadjacent to a promoter not naturally associated with the particularintroduced gene.

[0050] In preferred embodiments, the HAS or chondroitin synthaseencoding DNA segments further include DNA sequences, known in the artfunctionally as origins of replication or “replicons”, which allowreplication of contiguous sequences by the particular host. Such originsallow the preparation of extrachromosomally localized and replicatingchimeric segments or plasmids, to which HAS or chondroitin synthase DNAsequences are ligated. In more preferred instances, the employed originis one capable of replication in bacterial hosts suitable forbiotechnology applications. However, for more versatility of cloned DNAsegments, it may be desirable to alternatively or even additionallyemploy origins recognized by other host systems whose use iscontemplated (such as in a shuttle vector).

[0051] The isolation and use of other replication origins such as theSV40, polyoma or bovine papilloma virus origins, which may be employedfor cloning or expression in a number of higher organisms, are wellknown to those of ordinary skill in the art. In certain embodiments, theinvention may thus be defined in terms of a recombinant transformationvector which includes the HAS or chondroitin synthase coding genesequence together with an appropriate replication origin and under thecontrol of selected control regions.

[0052] Thus, it will be appreciated by those of skill in the art thatother means may be used to obtain the HAS or chondroitin synthase geneor cDNA, in light of the present disclosure. For example, polymerasechain reaction or RT-PCR produced DNA fragments may be obtained whichcontain full complements of genes or cDNAs from a number of sources,including other strains of Pasteurella or from eukaryotic sources, suchas cDNA libraries. Virtually any molecular cloning approach may beemployed for the generation of DNA fragments in accordance with thepresent invention. Thus, the only limitation generally on the particularmethod employed for DNA isolation is that the isolated nucleic acidsshould encode a biologically functional equivalent HA synthase.

[0053] Once the DNA has been isolated it is ligated together with aselected vector. Virtually any cloning vector can be employed to realizeadvantages in accordance with the invention. Typical useful vectorsinclude plasmids and phages for use in prokaryotic organisms and evenviral vectors for use in eukaryotic organisms. Examples includepKK223-3, pSA3, recombinant lambda, SV40, polyoma, adenovirus, bovinepapilloma virus and retroviruses. However, it is believed thatparticular advantages will ultimately be realized where vectors capableof replication in both Lactococcus or Bacillus strains and E. coli areemployed.

[0054] Vectors such as these, exemplified by the pSA3 vector of Dao andFerretti or the pAT19 vector of Trieu-Cuot, et al., allow one to performclonal colony selection in an easily manipulated host such as E. coli,followed by subsequent transfer back into a food grade Lactococcus orBacillus strain for production of HA or chondroitin. These are benignand well studied organisms used in the production of certain foods andbiotechnology products. These are advantageous in that one can augmentthe Lactococcus or Bacillus strain's ability to synthesize HA orchondroitin through gene dosaging (i.e., providing extra copies of theHAS or chondroitin synthase gene by amplification) and/or inclusion ofadditional genes to increase the availability of HA or chondroitinprecursors. The inherent ability of a bacterium to synthesize HA orchondroitin can also be augmented through the formation of extra copies,or amplification, of the plasmid that carries the HAS or chondroitinsynthase gene. This amplification can account for up to a 10-foldincrease in plasmid copy number and, therefore, the HAS or chondroitinsynthase gene copy number.

[0055] Another procedure that would further augment HAS or chondroitinsynthase gene copy number is the insertion of multiple copies of thegene into the plasmid. Another technique would include integrating theHAS or chondroitin synthase gene into chromosomal DNA. This extraamplification would be especially feasible, since the bacterial HAS orchondroitin synthase gene size is small. In some scenarios, thechromosomal DNA-ligated vector is employed to transfect the host that isselected for clonal screening purposes such as E. coli, through the useof a vector that is capable of expressing the inserted DNA in the chosenhost.

[0056] In certain other embodiments, the invention concerns isolated DNAsegments and recombinant vectors that include within their sequence anucleic acid sequence essentially as set forth in SEQ ID NO:1, 2, 3 or4. The term “essentially as set forth” in SEQ ID NO:1, 2, 3, or 4 isused in the same sense as described above and means that the nucleicacid sequence substantially corresponds to a portion of SEQ ID NO:1, 2,3 or 4 and has relatively few codons which are not identical, orfunctionally equivalent, to the codons of SEQ ID NO:1, 2, 3 or 4. Theterm “functionally equivalent codon” is used herein to refer to codonsthat encode the same amino acid, such as the six codons for arginine orserine, as set forth in Table I, and also refers to codons that encodebiologically equivalent amino acids.

[0057] It will also be understood that amino acid and nucleic acidsequences may include additional residues, such as additional—orC-terminal amino acids or 5′ or 3′ nucleic acid sequences, and yet stillbe essentially as set forth in one of the sequences disclosed herein, solong as the sequence meets the criteria set forth above, including themaintenance of biological protein activity where protein expression andenzyme activity is concerned. The addition of terminal sequencesparticularly applies to nucleic acid sequences which may, for example,include various non-coding sequences flanking either of the 5′ or 3′portions of the coding region or may include various internal sequences,which are known to occur within genes. Furthermore, residues may beremoved from the N or C terminal amino acids and yet still beessentially as set forth in one of the sequences disclosed herein, solong as the sequence meets the criteria set forth above, as well.

[0058] Allowing for the degeneracy of the genetic code as well asconserved and semi-conserved substitutions, sequences which have betweenabout 40% and about 80%; or more preferably, between about 80% and about90%; or even more preferably, between about 90% and about 99%; ofnucleotides which are identical to the nucleotides of SEQ ID NO:2 or 4will be sequences which are “essentially as set forth” in SEQ ID NO:2 or4. Sequences which are essentially the same as those set forth in SEQ IDNO:2 or 4 may also be functionally defined as sequences which arecapable of hybridizing to a nucleic acid segment containing thecomplement of SEQ ID NO:2 or 4 under standard or less stringenthybridizing conditions. Suitable standard hybridization conditions willbe well known to those of skill in the art and are clearly set forthherein.

[0059] The term “standard hybridization conditions” as used herein, isused to describe those conditions under which substantiallycomplementary nucleic acid segments will form standard Watson-Crickbase-pairing. A number of factors are known that determine thespecificity of binding or hybridization, such as pH, temperature, saltconcentration, the presence of agents, such as formamide and dimethylsulfoxide, the length of the segments that are hybridizing, and thelike. When it is contemplated that shorter nucleic acid segments will beused for hybridization, for example fragments between about 14 and about100 nucleotides, salt and temperature preferred conditions forhybridization will include 1.2-1.8×HPB at 40-50° C.

[0060] Naturally, the present invention also encompasses DNA segmentswhich are complementary, or essentially complementary, to the sequencesset forth in SEQ ID NO:2 or 4. Nucleic acid sequences which are“complementary” are those which are capable of base-pairing according tothe standard Watson-Crick complementarity rules. As used herein, theterm “complementary sequences” means nucleic acid sequences which aresubstantially complementary, as may be assessed by the same nucleotidecomparison set forth above, or as defined as being capable ofhybridizing to the nucleic acid segment of SEQ ID NO:2 or 4.

[0061] The nucleic acid segments of the present invention, regardless ofthe length of the coding sequence itself, may be combined with other DNAsequences, such as promoters, polyadenylation signals, additionalrestriction enzyme sites, multiple cloning sites, epitope tags, polyhistidine regions, other coding segments, and the like, such that theiroverall length may vary considerably. It is therefore contemplated thata nucleic acid fragment of almost any length may be employed, with thetotal length preferably being limited by the ease of preparation and usein the intended recombinant DNA protocol.

[0062] Naturally, it will also be understood that this invention is notlimited to the particular amino acid and nucleic acid sequences of SEQID NO:1, 2, 3, and 4. Recombinant vectors and isolated DNA segments maytherefore variously include the HAS or chondroitin synthase codingregions themselves, coding regions bearing selected alterations ormodifications in the basic coding region, or they may encode largerpolypeptides which nevertheless include HAS or chondroitinsynthase-coding regions or may encode biologically functional equivalentproteins or peptides which have variant amino acids sequences.

[0063] The DNA segments of the present invention encompass biologicallyfunctional equivalent HAS or chondroitin synthase proteins and peptides.Such sequences may arise as a consequence of codon redundancy andfunctional equivalency which are known to occur naturally within nucleicacid sequences and the proteins thus encoded. Alternatively,functionally equivalent proteins or peptides may be created via theapplication of recombinant DNA technology, in which changes in theprotein structure may be engineered, based on considerations of theproperties of the amino acids being exchanged. Changes designed by manmay be introduced through the application of site-directed mutagenesistechniques, e.g., to introduce improvements to the enzyme activity or toantigenicity of the HAS or chondroitin synthase protein or to test HASor chondroitin synthase mutants in order to examine HAS or chondroitinsynthase activity at the molecular level.

[0064] Traditionally, chemical or physical treatments of polysaccharideswere required to join two dissimilar materials. For example, a reactivenucleophile group of one polymer or surface was exposed to an activatedacceptor group of the other material. Two main problems exist with thisapproach, however. First, the control of the chemical reaction cannot berefined and differences in temperature and level of activation oftenresult in a distribution of several final products that vary from lot tolot preparation. For instance, several chains may be cross-linked in afew random, ill-defined areas and the resulting sample is nothomogenous. Second, the use of chemical reactions to join moleculesoften leaves an unnatural or nonbiological residue at the junction ofbiomaterials. For example, the use of an amine and an activated carboxylgroup would result in an amide linkage. This inappropriate residueburied in a carbohydrate may pose problems with biological systems suchas degradation products which accumulate to toxic levels or may triggeran immune response.

[0065] Most polysaccharide polymers must be of a certain length beforetheir physical or biological properties become apparent. Often thepolysaccharide must comprise at least 20-100 sugar units. Certainenzymes that react with exogenous polymers have been previouslyavailable, but typically add only one sugar unit. The unique enzymedescribed in the present invention, PmHAS, forms polymers of at least100-400 sugar units in length. The present invention thus results inlong, defined linear polymers composed of only natural glycosidiclinkages.

[0066] The two known glycosaminoglycan synthesizing enzymes fromPasteurella multocida bacteria normally make polymers similar to oridentical to vertebrate polymers. These bacteria employ thepolysaccharide, either HA (Type A bacteria) or chondroitin (Type Fbacteria), as an extracellular coating to serve as molecular camouflage.Native enzymes normally make polymer chains of a single type of sugarrepeat. If a recombinant HA synthase enzyme is employed, however, theenzyme can be forced to work on an exogenous acceptor molecule. Forinstance, the recombinant enzyme may be incubated with a polymeracceptor and the recombinant enzyme will then elongate the acceptor withUDP-sugar precursors. The known native enzymes do not perform thisreaction since they already contain a growing polymer chain.

[0067] PmHAS, a 972 amino acid residue protein from Pasteurellamultocida, is made in recombinant Escherichia coli. Other functionalderivatives of PmHAS, for example an enzyme called PmHAS-D, have beenproduced which are soluble. The soluble form can be prepared in largerquantities and in a purer state than the naturally -occurringfull-length enzyme. The preferred E. coli strains do not have an UDP-Glcdehydrogenase and therefore the recombinant enzyme does not make a HAchain in the foreign host. Therefore the enzyme is in a “virgin” statesince the empty acceptor site can be occupied with foreign polymers. Forexample, the recombinant enzyme may be incubated in a mixture containing50 mM Tris pH 7.2, 20 mM MnCl₂₁ 150-1600 mM UDP-GlcA, 200-1500 mMUDP-GlcNAc, and a suitable acceptor at 30° C. for 30-180 minutes.Suitable acceptors can be short HA chains (two or more sugar units) orshort chondroitin sulfate chains (5 sugar units) or long chondroitinsulfate chains (˜10² sugar units). In the case of the latter twoacceptors, the PmHAS, and its derivatives, then elongates the foreignacceptors (i.e. long or short chondroitan oligosaccharides) at theirnonreducing termini with authentic HA chains of up to 400 sugars. Thelength of the HA chain added onto the acceptor is controlled by alteringthe concentration of UDP-sugars and/or the reaction time. Immobilizedacceptors, such as beads or other solid objects with bound acceptoroligosaccharides, can also be extended by the PmHAS enzyme usingUDP-sugars. In this manner, the PmHAS enzyme can be used to attachpolysaccharide chains to any suitable acceptor molecule.

[0068] Type A P. multocida produces a HA capsule [GlcUA-GlcNAc repeats]and possesses the PmHAS enzyme. On the other hand, Type F P.multocidaproduce a chondroitan or chondroitan-like polymer capsule[GlcUA-GalNAc repeats]. The DNA encoding an open reading frame (GenBankaccession #AF195517) that is 87% identical to PmHAS at the protein levelhas been cloned; this new enzyme is called PmCS, the P. multocidachondroitan synthase. The amino acid sequence of PmCS is set forth inSeq ID NO: 3 and the PmCS nucleotide sequence is set forth in SEQ ID NO:4. As the PmCS enzyme's sequence is so similar to PmHAS, one of ordinaryskill in the art would be able to manipulate the PmCS in the same manneras that for PmHAS and any manipulation that was successful with regardto the PmHAS would be performable with the PmCS, with the exception thatchondroitan chains would be grafted instead of HA. Either HA orchondroitan chains can serve as acceptors for PmCS as both acceptorsserve well for PmHAS.

[0069] Such a hybrid polysaccharide material composed of both HA andchondroitin cannot be formed by any other existing process without (1)leaving unnatural residues and/or (2) producing undesirable crosslinkingreactions. The hybrid polysaccharide material can serve as abiocompatible molecular glue for cell/cell interactions in artificialtissues or organs and the HA/chondroitin hybrid mimics naturalproteoglycans that normally contain an additional protein intermediatebetween polymer chains. The present invention, therefore, obviates therequirement for a protein intermediary. A recombinant HA/chondroitinhybrid polysaccharide, devoid of such an intermediary protein, isdesirous since molecules from animal sources are potentiallyimmunogenic—the hybrid polysaccharide, however, would not appear as“foreign” to the host, thus no immune response is generated.

[0070] An intrinsic and essential feature of polysaccharide synthesis isthe repetitive addition of sugar monomer units to the growing polymer.The glycosyltransferase is expected to remain in association with thenascent chain. This feature is particularly relevant for HA biosynthesisas the HA polysaccharide product, in all known cases, is transported outof the cell; if the polymer was released, then the HAS would not haveanother chance to elongate that particular molecule. Three possiblemechanisms for maintaining the growing polymer chain at the active siteof the enzyme are immediately obvious. First, the enzyme possesses acarbohydrate polymer binding pocket or cleft. Second, the nascent chainis covalently attached to the enzyme during its synthesis. Third, theenzyme binds to the nucleotide base or the lipid moiety of the precursorwhile the nascent polymer chain is still covalently attached.

[0071] The HAS activity of the native PmHAS enzyme found in P. multocidamembrane preparations is not stimulated by the addition of HAoligosaccharides; theoretically, the endogenous nascent HA chaininitiated in vivo renders the exogenously supplied acceptor unnecessary.However, recombinant PmHAS produced in an E. coli strain that lacks theUDP-GlcUA precursor, and thus lacks a nascent HA chain, is able to bindand to elongate exogenous HA oligosaccharides. As mentioned above, thereare three likely means for a nascent HA chain to be held at or near theactive site. In the case of PmHAS, it appears that a HA-binding siteexists near or at the sugar transferase catalytic site.

[0072] Defined oligosaccharides that vary in size and composition areused to discern the nature of the interaction between PmHAS and thesugar chain. For example, it appears that the putative HA-polymerbinding pocket of PmHAS will bind and elongate at least an intact HAtrisaccharide (reduced tetramer). The monosaccharides GlcUA or GlcNAc,however, even in combination at high concentration, are not effectiveacceptors. Oligosaccharide binding to PmHAS appears to be somewhatselective because the heparosan pentamer, which only differs in theglycosidic linkages from HA-derived oligosaccharides, does not serve asan acceptor. However, chondroitan [GlcUA-GalNAc repeat] does serve as anacceptor for PmHAS.

[0073] To date, no other HA synthase besides PmHAS has been shown toutilize an exogenous acceptor or primer sugar. In an early study of acell-free HA synthesis system, preparations of native Group Astreptococcal HAS were neither inhibited nor stimulated by the additionof various HA oligosaccharides including the HA tetramer derived fromtesticular hyaluronidase digests. These membrane preparations wereisolated from cultures that were producing copious amounts of HApolysaccharide. The cells were hyaluronidase-treated to facilitatehandling. Therefore, it is quite likely that the native streptococcalenzyme was isolated with a small nascent HA chain attached to or boundto the protein much as suspected in the case of the native PmHAS.Theoretically, the existing nascent chain formed in vivo would block theentry and subsequent utilization of an exogenous acceptor by theisolated enzyme in vitro. With the advent of molecularly cloned HASgenes, it is possible to prepare virgin enzymes lacking a nascent HAchain if the proper host is utilized for expression.

[0074] Both heparin and chondroitin, in mammalian systems, aresynthesized by the addition of sugar units to the nonreducing end of thepolymer chain. In vivo, the glycosyltransferases initiate chainelongation on primer tetrasaccharides [xylose-galactose-galactose-GlcUA]that are attached to serine residues of proteoglycan core molecules. Invitro, enzyme extracts transfer a single sugar to exogenously addedheparin or chondroitin oligosaccharides; unfortunately, the subsequentsugar of the disaccharide unit is usually not added and processiveelongation to longer polymers does not occur. Therefore it is likelythat some component is altered or missing in the in vitro system. In thecase of heparin biosynthesis, it is postulated that a single enzymetransfers both GlcUA and GlcNAc sugars to the glycosaminoglycan chainbased on co-purification or expression studies.

[0075] Recent work with the E. coli K5 KfiC enzyme, which polymerizesheparosan, indicates that a single protein can transfer both sugars tothe nonreducing end of acceptor molecules in vitro. Processiveelongation, however, was not demonstrated in these experiments; crudecell lysates transferred a single sugar to defined even- or odd-numberedoligosaccharides. However, their initial mutagenesis experiments suggestthat at least two independent sites are involved in transfer of the twomonosaccharides.

[0076] Recombinant PmHAS adds single monosaccharides in a sequentialfashion to the nonreducing termini of the nascent HA chain. Elongationof HA polymers containing hundreds of sugars has been demonstrated invitro. The simultaneous formation of the disaccharide repeat unit is notnecessary for generating the alternating structure of the HA molecule.The intrinsic specificity and fidelity of each half-reaction (e.g. GlcUAadded to a GlcNAc residue or vice versa) apparently is sufficient tosynthesize authentic HA chains.

[0077] A great technical benefit resulting from the alternatingdisaccharide structure of HA is that the reaction can be dissected bycontrolling the availability of UDP-sugar nucleotides. By omitting orsupplying precursors in a reaction mixture, the glycosyltransferase maybe stopped and started at different stages of synthesis of theheteropolysaccharide. In contrast, there is no facile way to control ina step-wise fashion the glycosyltransferase enzymes that produceimportant homopolysaccharides such as chitin, cellulose, starch, andglycogen.

[0078] An alternative method for controlling polymerization has beenaccomplished by creating mutants that only add one sugar linkage onto ashort HA oligosaccharide. For example, PmHAS-E [PmHAS residues 1-650]can only add single GlcNAc sugars onto the non-reducing end (i.e. HAtetrasaccharide [GlcNAc-GlcUA-GlcNAc-GlcUA]) of an acceptor (i.e. formsthe HA pentamer). On the other hand, a mutant has been created andcalled PmHAS-D-D477N [PmHAS residues 1-703 with an asparaginesubstituted for the asparatate at position 477], which transfers only asingle GlcUA residue onto the non-reducing terminal GlcNAc group of theshort HA oligosaccharide. If extracts of two such mutants are mixedtogether with an acceptor in the presence of UDP-GlcNAc and UDP-GlcUA,then significant polymerization is achieved. It is also obvious that bycarrying out the steps of GlcNAc or GlcUA transfer separately andsequentially, almost any HA chain length should be possible. The same isalso true with regard to PmCS either alone or in combination with PmHAS.

[0079] As stated above, membrane preparations from recombinant E. colicontaining a PmHAS protein had HA synthase activity as judged byincorporation of radiolabel from UDP-[¹⁴C]GlcUA into polymer whenco-incubated with both UDP-GlcNAc and Mn ion. Due to the similarity atthe amino acid level of PmHAS to several lipopolysaccharidetransferases, it was hypothesized that HA oligosaccharides serve asacceptors for GlcUA and GlcNAc transfer. Addition of unlabeledeven-numbered HA tetramer (from testicular hyaluronidase digests) toreaction mixtures with recombinant PmHAS stimulates incorporation ofradiolabel from UDP-[¹⁴C]GlcUA into HA polymer by ˜20- to 60-fold incomparison to reactions without oligosaccharides as shown in FIG. 1.

[0080] In FIG. 1, a series of reactions containing PmHAS (30 μg totalmembrane protein) were incubated with UDP-[¹⁴C]GlcUA (2×10⁴ dpm, 120 μM)and UDP-GlcNAc (450 μM) in assay buffer (50 μl reaction vol) in thepresence of no added sugar (none) or various oligosaccharides (HA4, 4 μgHA tetramer; unsHA4/6, 4 μg unsaturated HA Δtetramer and Δhexamer;chito4, 50 μg chitotetraose; hep5, 20 μg heparosan pentamer). After 1hour, the reactions were analyzed by descending paper chromatography.Incorporation of radiolabel from UDP-[¹⁴C]GlcUA into high molecularweight HA is shown. Only intact tetramer (HA4) served as an acceptor.Reactions with heparosan and chitooligosaccharides, as well as GlcNAcand/or GlcUA (not shown), incorporated as much radiolabel as parallelreactions with no acceptor. The free monosaccharides GlcUA and GlcNAc,either singly or in combination at concentrations of up to 100 μM, donot serve as acceptors; likewise, the beta-methyl glycosides of thesesugars do not stimulate HAS activity.

[0081] In the same manner, PmHAS has been shown to add sugars onto achondroitan pentamer acceptor. The PmHAS and reagents were prepared inthe same manner as shown in FIG. 1, except that a chondroitan pentamerwas used as the acceptor molecule. The results of this experiment areshown in TABLE A. TABLE A Incorporation of ¹⁴C- Sugar mass GlcUA dpmnone — 60 HA₄  5 μg 2,390 Chondroitan Pentamer 20 μg 6,690

[0082] Thus, it can be seen that the PmHAS can utilize numerousacceptors or primer molecules as the basis for forming a polysaccharidepolymer chain.

[0083] The activity of recombinant PmHAS is dependent on thesimultaneous incubation with both UDP-sugar precursors and a Mn²⁺ ion.The level of incorporation is dependent on protein concentration, on HAoligosaccharide concentration, and on incubation time as shown in FIG.2. In FIG. 2, two parallel reactions containing PmHAS with even-numberedHA oligosaccharides (105 μg membrane protein/point with a mixture of HAhexamer, octamer, and decamer, 4.4. μg total; solid circles) or six-foldmore PmHAS without oligosaccharide acceptor (630 μg protein/point; opencircles) were compared. The enzyme preparations were added to prewarmedreaction mixtures containing UDP-[¹⁴C]GlcUA (240 μM 6×10⁴ dpm/point) andUDP-GlcNAc (600 μM) in assay buffer. At various times, 50 μl aliquotswere withdrawn, terminated, and analyzed by paper chromatography. Theexogenously supplied acceptor accelerated the bulk incorporation ofsugar precursor into polymer product by PmHAS, but the acceptor was notabsolutely required.

[0084] HA synthesized in the presence or the absence of HAoligosaccharides is sensitive to HA lyase (>95% destroyed) and has amolecular weight of ³1-5′ 10⁴ Da (˜50-250 monosaccharides). Norequirement for a lipid-linked intermediate was observed as neitherbacitracin (0.5 mg/ml) nor tunicamycin (0.2 mg/ml) alter the level ofincorporation in comparison to parallel reactions with no inhibitor.

[0085] Gel filtration chromatography analysis of reactions containingrecombinant PmHAS, ³H-HA tetramer, UDP-GlcNAc and UDP-GlcUA show thatlabeled polymers from ˜0.5 to 5′ 10⁴ Da (25-250 monosaccharides) aremade as shown in FIG. 3. In FIG. 3, gel filtration analysis on SephacrylS-200 (20 ml column, 0.7 ml fractions) shows that PmHAS-D makes HApolysaccharide using HA tetramer acceptor and UDP-sugars. Dextrans ofgreater than or equal to 80 kDa (˜400 monosaccharides) elute in the voidvolume (Vo arrow). The starting tetramer elutes in the included volume(Vi arrow). Membranes (190 μg total protein), UDP-GlcUA (200 μM),UDP-GlcNAc (600 μM), and radiolabeled ³H-HA tetramer (1.1×10⁵ dpm) wereincubated for 3 hours before gel filtration (solid squares). As anegative control, a parallel reaction containing all the componentsexcept for UDP-GlcNAc was analyzed (open squares). The small primer waselongated into higher molecular weight product if both precursors weresupplied. In a parallel reaction without UDP-GlcNAc, the elution profileof the labeled tetramer is not altered.

[0086] The activity of the native PmHAS from P. multocida membranes,however, is not stimulated by the addition of HA oligosaccharides undersimilar conditions. The native PmHAS enzyme has an attached or boundnascent HA chain that is initiated in the bacterium prior to membraneisolation. The recombinant enzyme, on the other hand, lacks such anascent HA chain since the E. Coli host does not produce the UDP-GlcUAprecursor needed to make HA polysaccharide. Therefore, the exogenousHA-derived oligosaccharide has access to the active site of PmHAS andcan be elongated.

[0087] The tetramer from bovine testicular hyaluronidase digests of HAterminates at the nonreducing end with a GlcUA residue and this moleculeserved as an acceptor for HA elongation by PmHAS. On the other hand, theDtetramer and Dhexamer oligosaccharides produced by the action ofStreptomyces HA lyase did not stimulate HA polymerization as shown inFIG. 1; “unsHA4/6”. As a result of the lyase eliminative cleavage, theterminal unsaturated sugar is missing the C4 hydroxyl of GlcUA whichwould normally be extended by the HA synthase. The lack of subsequentpolymerization onto this terminal unsaturated sugar is analogous to thecase of dideoxynucleotides causing chain termination if present duringDNA synthesis. A closed pyranose ring at the reducing terminus was notrequired by PmHAS since reduction with borohydride did not affect the HAtetramer's ability to serve as an acceptor thus allowing the use ofborotritide labeling to monitor the fate of oligosaccharides.

[0088] Neither recombinant Group A HasA nor recombinant DG42 producedelongated HA-derived oligosaccharides into larger polymers in yeast.First, the addition of HA tetramer (or a series of longeroligosaccharides) did not significantly stimulate nor inhibit theincorporation of radiolabeled UDP-sugar precursors into HA (³±5% ofcontrol value). In parallel experiments, the HAS activity of HasA orDG42 was not affected by the addition of chitin-derivedoligosaccharides. Second, the recombinant enzymes did not elongate theradiolabeled HA tetramer in the presence of UDP-sugars (Table II). Thesesame preparations of enzymes, however, were highly active in theconventional HAS assay in which radiolabeled UDP-sugars were polymerizedinto HA. TABLE II Incorporation of HA4 into polymer Enzyme Units^(a)EDTA (pmoles) PmHAS    6^(b) − 240 + 1.7 HasA  9,800 − ≦0.2 + ≦0.2 DG4211,500 − ≦0.1 + ≦0.3

[0089] As shown in Table II, the various recombinant enzymes were testedfor their ability to convert HA tetramer into molecular weight products.The reactions contained radiolabeled HA tetramer (5-8×10⁵ dpm), 750 μMUDP-GlcNAc, 360 μM UDP-GlcUA, 20 mM XCI₂, 50 mM Tris, pH 7-7.6 (therespective X cation and pH values used for each enzyme were: PmHAS,Mn/7.2; Xenopous DG42, Mg/7.6; Group A streptococcal HasA, Mg/7.0), andenzyme (units/reaction listed). As a control, parallel reactions inwhich the metal ion was chelated (22 mM ethylenediaminetetraacetic acidfinal; EDTA column, rows with +) were tested; without free metal ion,the HAS enzymes do not catalyze polymerization. After 1 hour incubation,the reactions were terminated and subjected to descending paperchromatography. Only PmHAS-D could elongate HA tetramer even though allthree membrane preparations were very active in the conventional HASassay (incorporation of [¹⁴C]GlcUA from UDP-GlcUA into polymer whensupplied UDP-GlcNAc).

[0090] Thin layer chromatography was utilized to monitor thePmHAS-catalyzed elongation reactions containing ³H-labeledoligosaccharides and various combinations of UDP-sugar nucleotides. FIG.4 demonstrates that PmHAS elongated the HA-derived tetramer by a singlesugar unit if the next appropriate UDP-sugar precursor was available inthe reaction mixture. GlcNAc derived from UDP-GlcNAc was added onto theGlcUA residue at the nonreducing terminus of the tetramer acceptor toform a pentamer. On the other hand, inclusion of only UDP-GlcUA did notalter the mobility of the oligosaccharide. If both HA precursors aresupplied, various longer products are made. In parallel reactions,control membranes prepared from host cells with a vector plasmid did notalter the mobility of the radiolabeled HA tetramer under anycircumstances. In similar analyses monitored by TLC, PmHAS did notutilize labeled chitopentaose as an acceptor.

[0091] As shown in FIG. 4, PmHAS extended an HA tetramer. In FIG. 4,radiolabeled HA tetramer (HA4 8×10³ dpm ³H) with a GlcUA at thenonreducing terminus was incubated with various combinations ofUDP-sugars (A, 360 μM UDP-GlcUA; N, 750 μM UDP-GlcNAc; 0, no UDP-sugar),and PmHAS (55 μg membrane protein) in assay buffer for 60 minutes. Thereactions (7 μl total) were terminated by heating at 95 degrees Celsiusfor 1 minute and clarified by centrifugation. Portions (2.5 μl) of thesupernatant were spotted onto the application zone of a silica TLC plateand developed with solvent (1.25:1:1 butanol/acetic acid/water). Thebeginning of the analytical layer is marked by an arrow. The positionsof odd-numbered HA oligosaccharides (S lane) are marked as number ofmonosaccharide units. This autoradiogram (4 day exposure) shows thesingle addition of a GlcNAc sugar onto the HA tetramer acceptor to forma pentamer when only the subsequent precursor is supplied (N). Themobility of the labeled tetramer is unchanged if only the inappropriateprecursor, UDP-GlcUA (A), or no UDP-sugar (O) is present. If bothUDP-sugars are supplied, then a ladder of products with sizes of 5, 7,9, 11, and 13 sugars is formed (+AN). In a parallel experiment,chitopentaose (8×10⁴ dpm ³H) was tested as an acceptor substrate. Underno condition was this structurally related molecule extended by PmHAS.

[0092] HA-derived oligosaccharides with either GlcUA or GlcNAc at thenonreducing terminus served as acceptors for PmHAS (FIG. 5). In FIG. 5,radiolabeled HA pentamer (HA5, 5×10³ dpm ³H) or HA tetramer (HA4, 25×10³dpm ³H) was incubated with PmHAS and various combinations of UDP-sugars(as in FIG. 4) for 2 or 20 minutes. Portions (1.5 μl) of the supernatantwere spotted onto the TLC plate and developed in 1.5:1:1 solvent. Thisautoradiogram (1 mo. exposure) shows the single addition of a sugar ontoan acceptor when only the appropriate precursor is supplied (HA4, N laneand HA5, A lane). If both UDP-sugars are supplied (+AN lanes), then aladder of products with final sizes of 6, 8, and 10 sugars is formedfrom either HA4 or HAS in 2 minutes. After 20 minutes, a range of odd-and even-numbered product sugars are observed in reactions with HA4 andboth UDP-sugars. In the 20 minute reaction with HA5 and both UDP-sugars,the HA products are so large that they do not migrate from theapplication zone.

[0093] Within two minutes, 2 to 6 sugar units were added, and after 20minutes, 9 to ³15 units were added. In the experiments with the HAtetramer and both sugars, a ladder of even- and odd-numbered products isproduced at the 20 minute time point. Therefore, in combination with theresults of the single UDP-sugar experiments, the PmHAS enzyme transfersindividual monosaccharides sequentially during a polymerizationreaction.

[0094] 1. HA Synthase Isolation and Assays—Membrane preparationscontaining recombinant PmHAS (GenBank AF036004) were isolated from E.coli SURE(pPmHAS). Membrane preparations containing native PmHAS wereobtained from the P. multocida strain P-1059 (ATCC #15742). PmHAS wasassayed in 50 mM Tris, pH 7.2, 20 mM MnCl₂, and UDP-sugars(UDP-[¹⁴C]GlcUA, 0.3 Ci/mmol, NEN and UDP-GlcNAc) at 30° C. The reactionproducts were analyzed by various chromatographic methods as describedbelow. Membrane preparations containing other recombinant HAS enzymes,Group A streptococcal HasA or Xenopus DG42 produced in the yeastSaccharomyces cerevisiae, were prepared.

[0095] 2. Acceptor Oligosaccharides—Uronic acid was quantitated by thecarbazole method. Even-numbered HA oligosaccharides [(GlcNAc-GlcUA)_(n)]were generated by degradation of HA (from Group A Streptococcus) witheither bovine testicular hyaluronidase Type V (n=2-5) or Streptomyceshyaluroniticus HA lyase (n=2 or 3) in 30 mM sodium acetate, pH 5.2, at30° C. overnight. The latter enzyme employs an elimination mechanism tocleave the chain resulting in an unsaturated DGlcUA residue at thenonreducing terminus of each fragment. For further purification anddesalting, some preparations were subjected to gel filtration with P-2resin (BioRad) in 0.2 M ammonium formate and lyophilization.Odd-numbered HA oligosaccharides [GlcNAc(GlcUA-GlcNAc)_(n)] ending in aGlcNAc residue were prepared by mercuric acetate-treatment of partial HAdigests generated by HA lyase (n=2-7). The masses of the HAoligosaccharides were verified by matrix-assisted laser desorptionionization time-of-flight mass spectrometry. Sugars in water were mixedwith an equal volume of 5 mg/ml 6-azo-2-thiothymine in 50%acetonitrile/0.1% trifluoroacetic acid, and rapidly air-dried on thetarget plate. The negative ions produced by pulsed nitrogen laserirradiation were analyzed in linear mode (20 kV acceleration; PerceptiveVoyagera).

[0096] Other oligosaccharides that are structurally similar to HA werealso tested in HAS assays. The structure of heparosan pentamer derivedfrom the E. coli K5 capsular polysaccharide isb(1,4)GlcNAc-a(1,4)GlcUA]₂-b(1,4)GlcNAc; this carbohydrate has the samecomposition as HA but the glycosidic linkages between themonosaccharides are different. The chitin-derived oligosaccharides,chitotetraose and chitopentaose, are b(1,4)GlcNAc polymers made of 4 or5 monosaccharides, respectively.

[0097] Various oligosaccharides were radiolabeled by reduction with 4 to6 equivalents of sodium borotritide (20 mM, NEN; 0.2 Ci/mmol) in 15 mMNaOH at 30° C. for 2 hrs. ³H-oligosaccharides were desalted on a P-2column in 0.2 M ammonium formate to remove unincorporated tritium andlyophilized. Some labeled oligosaccharides were further purifiedpreparatively by paper chromatography with Whatman 1 developed inpyridine/ethyl acetate/acetic acid/H₂O (5:5:1:3) before use as anacceptor.

[0098] 3. Chromatographic Analyses of HA Synthase ReactionProducts—Paper chromatography with Whatman 3M developed in ethanol/1Mammonium acetate, pH 5.5 (65:35) was used to separate high molecularweight HA product (which remains at the origin) from UDP-sugars andsmall acceptor oligosaccharides. In the conventional HAS assay,radioactive UDP-sugars are polymerized into HA. To obtain the sizedistribution of the HA polymerization products, some samples were alsoseparated by gel filtration chromatography with Sephacryl S-200(Pharmacia) columns in 0.2 M NaCl, 5 mM Tris, pH 8. Columns werecalibrated with dextran standards. The identity of the polymer productswas assessed by sensitivity to specific HA lyase and the requirement forthe simultaneous presence of both UDP-sugar precursors during thereaction. Thin layer chromatography [TLC] on high performance silicaplates with application zones (Whatman) utilizing butanol/aceticacid/water (1.5:1:1 or 1.25:1:1) development solvent separated³H-labeled oligosaccharides in reaction mixes. Radioactive moleculeswere visualized after impregnation with EnHance spray (NEN) andfluorography at −80° C.

[0099] An anti-PmHAS monospecific antibody reagent has also beenidentified that routinely monitors the protein by Western blots orimmunoassays; this reagent can be used to normalize protein expressionlevels. The DNA inserts encoding the enzyme sequence from interestingmutants picked up in screens can be subcloned and completely sequencedto verify and to identify the mutation site.

[0100] A series of truncated versions of PmHAS (normally a 972-residuemembrane protein) were created which produce proteins with alteredphysical properties (i.e. proteins that are more conducive to high-levelexpression and purification) and altered function (i.e. singletransferase activity). Polymerase chain reaction [PCR] was used toamplify a portion of the PmHAS gene using a primer corresponding to theauthentic N-terminus sequence and a primer corresponding to an internalcoding region which ended in a stop codon. The coding regions for thetruncated proteins were cloned into an Escherichia coli expressionplasmid (pKK223-3; Pharmacia) under control of the tac promoter. The DNAsequence was verified by automated sequencing.

[0101] The truncation series was generated and tested for activity. Allproteins were made at the expected molecular weight, but not allproteins were active. TABLE III Name Residues of PmHAS Activity PmHAS-A437-972 N.D. PmHAS-B 437-756 N.D. PmHAS-C  1-756 HA Synthase PmHAS-D 1-703 HA Synthase PmHAS-E  1-650 GlcNAc Transferase PmHAS-F 152-756N.D.

[0102] Analysis of induced cell cultures containing the plasmid with a703-residue open reading frame revealed that a new 80-kDa protein, namedPmHAS-D, was produced in large quantities. Furthermore, functionalPmHAS-D was present in the soluble fraction of the cell lysate; thusallowing for rapid extraction and assay of the enzyme. PmHAS-D waspurified by sequential chromatography steps shown in FIG. 6. In FIG. 6,a soluble, active form of the HA synthase was constructed with molecularbiological techniques. The recombinant enzyme from E. coli was purifiedby conventional chromatography with yields of up to 20 mg/liter of cellculture. FIG. 6 is a stained electrophoretic gel loaded with samples ofPmHAS-D (marked with a star) during different stages of chromatography.This catalyst (and improved mutant versions) can be used to prepare HAcoatings on artificial surfaces or HA extensions on suitable acceptormolecules.

[0103] The PmHAS-D is highly active and at least 95% pure as assessed bydenaturing polyacrylamide gel electrophoresis. Mass spectrometricanalysis indicates that the PmHAS-D is the desired protein due to theclose agreement of the calculated and the observed mass values. A buffersystem has also been developed to stabilize the enzymatic activity inthe range of 0° to 37° C.

[0104] Site-directed mutagenesis was then used to prepare versions ofPmHAS-D with altered enzymatic activity. Synthetic DNA oligonucleotidesand multiple rounds of extension with Pfu DNA polymerase were used toadd mutations to the coding region using the Quick-Change system fromStratagene. Through use of primers with mixed bases at certainpositions, a wide variety of amino acid changes were generated. DNAsequencing was then employed to identify the changed residue. SeveralPmHAS-D mutants have also been obtained having altered sugar transferaseactivity. Similar methodology has also been used to alter theHA-acceptor binding site of PmHAS-D.

[0105] Two positions of the PmHAS-D sequence were mutated in the initialtrials. Conserved aspartates at residue 196 or 477 were critical for HASactivity. TABLE IV Mutation (*) HAS Activity GlcNActase GlcUAtase D196EW/O W/O YES D196N W/O W/O YES D196K W/O W/O YES D477E W/O YES W/O D477NW/O YES W/O D477K W/O YES W/O WILD TYPE YES YES YES CONTROL

[0106] The mutant enzymes are useful for adding on a single GlcNAc or asingle GlcUA onto the appropriate acceptor oligosaccharide. It appearsthat PmHAS has two domains or two modules for transferring each sugar.One of ordinary skill in the art, given this specification, would beable to shift or to combine various domains to create new polysaccharidesynthases capable of producing new polysaccharides with alteredstructures. Within such use, a variety of grafting techniques arisewhich utilize PmHAS as the prototype. A graphical representation of eachmutant as it relates to the PmHAS-D sequence, is shown in FIG. 7.

[0107]FIG. 8 is a graphical representation of a mutant combinationassay. HAS enzyme assays were performed in the presence of wild typePmHAS alone, D196 mutant alone, D477 mutant alone, or in the presence ofboth D196 and D477 mutants. Equal amounts of each enzyme were testedwith a small amount of HA acceptor sugar in the typical reaction bufferat 30 degrees Celsius. Two time points were measured (cross-hatched, 25minutes; black, 1.5 hours) for each assay. The two mutants work togetherto make HA polymer; by itself, a single mutant cannot make HA polymer.

[0108] Enzyme activity of the PmHAS-D mutants is shown in FIG. 9.Extracts of the mutants were used for all three kinds of assays: for HApolymer production, for GlcUA-Tase activity and for GlcNAc-Taseactivity. Equivalent amounts of PmHAS-D proteins (based on Western blotanalysis) were assayed. The activities were indicated as the percentageof the activity of wild type PmHAS-D.

[0109] With the advent of new biomaterials and biomimetics, hybridpolysaccharide materials will be required to serve the medical field. Amajor goal of bioengineering is the design of implanted artificialdevices to repair or to monitor the human body. Versatilesemiconductors, high-strength polymers, and durable alloys have manyproperties that make these materials desirable for bioengineering tasks.However, the human body has a wide range of defenses and responses thathinder the utilization of modern man-made substances. As differenttissues and organs are identified as future recipients of biotechnology,it will be imperative to have an assortment of non-immunogenic polymersthat can act as adhesives or protective coatings. Emulsification oradhesion industrial processes are also well suited for use with thepresent invention and other more suitable enzymes may be employed tograft useful molecules.

[0110] Chemical sensors which utilize electrochemical reactions havepromise in many biomedical applications. In particular, the measurementof blood glucose for home monitoring of diabetics is of great interest.Unfortunately, biochemical sensors for glucose and other biologicalchemicals have not achieved their anticipated level of success. Problemswith sensor reliability, selectivity, and material stability havedelayed the fruition of the biosensor market. New methods to depositselective materials onto electronic substrates while maintainingcompatibility with biological systems are needed. The present inventionprovides such a method. Through the use of the PmHAS-D enzyme, anelectronic or metallic substrate which has been primed with a suitableexogenous HA oligosaccharide can be coated with a layer of HA. Such alayer of HA would protect the electronic substrate from the biologicalimmune systems while allowing full function of the electronic ormetallic material.

[0111] Presently, commercially available glucose sensors operate throughthe electrochemical oxidation and reduction of glucose oxidase found ina patient's blood. Typically the patient must prick their finger severaltimes daily to obtain the blood sample needed for the sensor. Once inthe sensor, the glucose oxidase reacts with glucose to form gluconicacid. The reduced form of the enzyme reacts with an electron mediatorsuch as ferricyanide to form ferrocyanide. A sensor electrode oxidizesthe ferrocyanide creating a current proportional to the concentration ofglucose in the blood.

[0112] As with many biosensors, a significant shift toward continualmonitoring using minimally invasive or implantable sensing devices,which require fully integrated microelectronic capabilities whilemaintaining biocompatibility, remains a future trend in glucose sensordevelopment. A glucose microsensor using microfabrication of sensorarrays is a convenient means of implantation and has a high sensitivitythreshold. Presently, no commercial glucose microsensor exists. Issuessuch as sensor selectivity and stability have hindered the developmentof an implantable glucose microsensor. Because of the harsh environmentof the human body, biocompatibility becomes an important issue to thestability and reliability of the biosensor. Those working in the arthave looked at a variety of polymer membranes that protect the sensorfrom the body. Some have also chemically attached electron mediators andenzymes directly to polymer materials thereby providing electricalconnection and improved stability and safety of the sensor for in vivouse. A means of incorporating biological materials to the sensingsurface while maintaining sensing function would be beneficial. Thepresent invention provides such a method for producing non-immunogeniccoating for sensors as well as other biomaterials.

[0113] In the present invention, HA oligosaccharides and other novelprimer materials are deposited onto the inorganic substrate usingchemistry known to those of ordinary skill in the art and similarreaction processes. For example, a reactive epoxy surface can be madewhich in turn can react with amino compounds derived fromHA-oligosaccharides. Once the primer materials have been deposited ontothe inorganic substrate, PmHAS-D is utilized to form a protectivecoating of HA-polymer on the inorganic substrate. The HA polymer coatingthereby protects the substrate from the body's immune system whileallowing the substrate to perform an indicated purpose such as sensing,detection or drug delivery.

[0114] The majority of existing artificial materials suitable forimplants and sensors, to some degree, usually (a) cause a foreign-bodyreaction due to the interactions with tissues or biological fluids or(b) lack substantial connectivity with the body due to their relativeinertness. The HA polymer coating of the present invention overcomesthese two stumbling blocks. A uniform coating of naturally occurring HAprevents an artificial components implanted into the body from spawningadverse effects such as an immune response, inappropriate clottingand/or inflammation. Furthermore, because HA is involved in maintainingthe integrity of tissues and wound-healing, the HA polysaccharidecoating encourages the acceptance of the artificial structure within thebody.

[0115] The HA polymer attached to a biosensor acts as an externalbarrier protecting the sensor from the body's environment. However, inany sensing application, the chemical analyte must be able to contactthe sensing material. Therefore, the HA polymer layer must allowtransport of glucose to regions inside the sensor. Other molecules alsoexist in the blood that may interfere with the sensor response. Phaseequilibrium between components in the blood and the HA polymer layerdetermine the local environment of the sensing layer. The transportproperties of thin HA polymer layers also allow for the use of the HApolymer as a packaging material. The HA polymer outer coating allowstransport of the glucose analyte in a diffusion-controlled manner whilepreventing biological materials from damaging the electronic device. Asthe HA polymer to be deposited consists of tangled, linear chains ofhydrophilic sugars, glucose and other small compounds move relativelyfreely in the layer. On the other hand, medium to large proteins, whichmay foul the sensor, are excluded from the HA layer.

[0116] As stated previously, there is precedent for utilizing HA in themedical treatment of humans. Currently, HA is employed in eye surgery,joint fluid replacement, and some surgical aids. Much investigation onthe use of HA to coat biomedical devices is also underway. In thepreviously described coating methods, HA extracted from animal orbacterial sources is typically chemically crosslinked or physicallyadsorbed onto a surface. Potential problems with these methodologiesinclude: (a) immunoreaction with animal-borne contaminants and/orintroduced chemical crosslinking groups and (b) the lack ofreproducibility of the coating configuration. In the present invention,HA polymer chains are produced in situ using the purified biosyntheticenzyme, PmHAS-D. (FIG. 10). In FIG. 10, the schematic representation of1^(st) generation HA coating on silicon is shown. A silane and then asugar primer are attached to the silicon surface. PmHAS-D then elongatesthe primer with appropriate sugars to form a biocompatible coating. Thelength of the HA polymer (100 to 103 sugars) are adjusted to fit theparticular coating application.

[0117] Due to the relative absence of foreign components or artificialmoieties, no immunological problems occur. Depending on the particularapplication, the polymer length and the chain orientation can becontrolled with precision. The polysaccharide surface coatings of thepresent invention improves the biocompatibility of the artificialmaterial, lengthens the lifetime of the device in the cellularenvironment, and encourages natural interactions with host tissues.

[0118] With regard to surface coatings on solid materials,polyacrylamide beads have been coated with the HA polymer using PmHAS-Das the catalyst. First, aminoethyl-beads were chemically primed with HAoligosaccharide (a mixture of 4, 6, and 8 sugars long) by reductiveamination. Beads, HA oligosaccharide, and 70 mM NaCNBH₄ in 0.2 M boratebuffer, pH 9, were incubated at 42° C. for 2 days. The beads were washedwith high and low salt buffers before use in the next step. Controlbeads without priming sugar or with chitopentaose [(GlcNAc)₅] were alsoprepared; beads without HA would not be expected to prime HA synthesisand the chitopentaose does not serve as an acceptor for PmHAS. Second,the various preparations of beads (15 p liters) were incubated withPmHAS-D (3 μg), 150 mM UDP-[³H]GlcNAc, 60 mM UDP-[¹⁴C]GlcUA, 20 mMMnCl₂, in 50 mM Tris, pH 7.2, at 30° C. for 60 min. The beads were thenwashed with high and low salt buffers. Radioactivity linked to beads(corresponding to the sugars) was then measured by liquid scintillationcounting Table V. TABLE V Bound GlcUA Bound GlcNAc Bead Type EnzymeAdded? (¹⁴C dpm) (³H dpm) HA primer yes 990 1140 HA primer no 10 10Chito primer yes 24 18 No primer yes 5 35

[0119] Only HA beads primed with the HA oligosaccharide and incubatedwith PmHAS-D incorporated the radiolabel from both UDP-sugar precursorsindicating that the short HA sugar attached to the bead was elongatedinto a longer HA polymer by the enzyme. Thus far, no other known HAsynthase possesses the desired catalytic activity to apply an HA polymercoating onto a primed substrate.

[0120] Thus, as shown above, an authentic HA oligosaccharide primer waschemically coupled to a polyacrylamide surface and then this primer wasfurther elongated using the PmHAS enzyme and UDP-sugars. Depending onthe substrate, the reaction conditions can be optimized by one ofordinary skill in the art. For example, the mode of semiconductormodification, buffer conditions, HA elongation reaction time, andstoichiometry can be varied to take into account any single or multiplereaction variation. The resulting coatings can then be evaluated forefficacy and use.

[0121] In order to scale-up and to facilitate the biocompatible HAcoating process to a level practical for medical devices in the future,(a) a new synthetic molecule that would substitute for the HAoligosaccharide with the original PmHAS-D enzyme will be used; or (b) amutant form of the PmHAS-D enzyme that will utilize a “simpler” organicmolecule as the primer will be used.

[0122] The critical structural elements of the HA oligosaccharideacceptor or primer molecule are currently being tested and identified.The smallest acceptor molecule with activity tested thus far is an HAtetramer [non-reducing -GlcUA-GlcNAc-GlcUA-GlcNAc- reducing]. Recentdata suggests that the PmHAS-D enzyme has some flexibility with respectto the identity of the hexosamine group; i.e. other isomers willsubstitute for the GlcNAc sugar. For example, chondroitan pentamer[GalNAc-GlcUA-GalNAc-GlcUA-GalNAc], serves as an effective acceptor forrecombinant PmHAS. Therefore, a synthetic molecule consisting of severalhydroxyl groups, a pair of negatively charged groups (corresponding tothe carboxyl groups of GlcUA sugar), and hydrophobic patches (analog ofthe carbon-rich side of the sugar ring) may work as a primer. Such anapproach is not unprecedented as the polymerization of heparin, aglycosaminoglycan, can be primed with a rather simple aromatic xylosideinstead of a complex proteoglycan core.

[0123] Computer modeling of HA oligosaccharides can visualize potentialmolecular shape. However, some proteins distort the sugar chains uponbinding, thus making computer modeling somewhat more complicated. Themost efficacious method of finding an artificial primer is acombinatorial chemistry approach. Closely related series of moleculesare screened by high-throughput assay methodologies in order to detectHA elongation. Native PmHAS-D is then tested for the ability to add anHA polymer onto synthetic primer candidates in a typical 96-well plateformat. For example, a series of synthetic peptides (6 to 8 residues)terminating with a GlcNAc group using conventional F^(moc) chemistry canbe generated. Such peptides are particularly promising because they canadopt a variety of conformations and fit within the PmHAS-D HA-bindingpocket via an induced fit mechanism. Synthetic peptide chemistry is alsomuch less cumbersome than carbohydrate chemistry. One of ordinary skillin the art, given the present specification, would be capable of usingthe known synthetic peptide chemistry techniques.

[0124] The amino acids are chosen with the goal of mimicking theproperties of the GlcNAcGlcUA sugar repeats of HA. For example, use ofglutamate or asparatate as a substitute for the acid group of GlcUA, oruse of glutamine or asparagine as a substitute for the amide group ofGlcNAc. Serine, threonine, or tyrosine can be used as substitutes forthe hydroxyl groups and sugar rings in general. The peptide libraryterminates with a GlcNAc sugar group so that the demands on the PmHAS-Denzyme's binding site and catalytic center are not overly burdensome. Avast variety of distinct peptides are made in parallel with acombinatorial approach; for example, with a hypothetical 6-7 residuepeptide containing 1 to 3 different amino acids at each position, thereare hundreds of possible peptides. The peptide combinatorial librarieswill either be immobilized on plastic pins or plates.

[0125] The present invention also encompasses the development of amutant version of PmHAS-D that will utilize a simpler molecule than anHA oligosaccharide as a primer. Chitopentaose (β1,4-GlcNAc homopolymer)is one such potential variant primer. Native PmHAS-D does not utilizechitopentaose as a primer, but a mutant PmHAS-D may potentially elongatechitopentaose, a more readily available substance. The chitopentaoseprimer is attached to the solid phase by reductive amination to anamino-containing plate or to a carrier protein (albumin) forimmobilization on a normal plastic plate. Various mutants could then bescreened for function. Other potential non-sugar mimics contemplated foruse are short poly(ethleneglycol)-based copolymers containing styrene,sulfonate, acrylate, and/or benzoate groups.

[0126] Photoaffinity labeling is used to cross-link a radioactive HAoligosaccharide analog containing an aryl azide to the PmHAS-D protein.The binding site of the PmHAS-D protein is obtained through peptidemapping and Edman sequencing. With this information, mutants areprepared with alterations at the binding site. In the chitopentaoseexample, removal of some of the basic residues of the HA-binding site(which normally contact the carboxylate of GlcUA) and substitution ofneutral polar residues would be chosen. As described above, a variety ofsite-directed mutants using a mutagenic oligonucleotide with mixed basesat certain positions have been generated. Such a mixed-base approacheconomizes on the number of custom oligonucleotides and transformationsrequired. A high-throughput screen is then used to assess the ability ofthe mutant PmHASs to elongate the synthetic primer with a HA chain. Anempirical approach can also be used randomly mutate PmHAS (eitherchemical mutagens or with a passage through a mutator strain) and thenscreen.

[0127] An assay has been designed to measure successful HA elongationreactions in a 96-well format (FIG. 11). The assay is shown in FIG. 11in a graphical representation. Utilizing this assay many mutants can bescreened in parallel. This screening method is facilitated by the factthat (i) a protocol to readily extract functional recombinant PmHAS-Dfrom E. coli cultures in a 96-well plate format with minimal processingexists and (ii) sensitive methods to detect HA on solid-phase microtiterplates exists. Cultures and extracts are transferred in parallel withmulti-channel pipettes. HAS activity produced by 10-30 μl of inducedcell culture (with an absorbance=1 at 600 nm) is routinely detected andthe wells have a working volume of 200-300 μl, thus multiple assays ordetection of low HA production is possible. Other components in the celllysate do not interfere with the HAS assay. The extracts are stable at−80° C. for long-time storage. For detection of HA elongation,specificity of a HA-binding protein probe [HABP], biotinylated aggrecan,is capitalized upon. This probe binds elongated HA chains with highaffinity but not small HA primers (4-6 sugars long). The bound HABPprobe is detected by virtue of the biotin tag that is measured withfluorescent, radiolabeled, or enzyme-conjugated avidin (a biotin-bindingprotein).

[0128] In order to identify enzymes with low activities or reactionswith poor primers, radioactive sugar incorporation (from UDP-[³H]GlcNACor UDP-[¹⁴C]GlcUA) is measured instead of using the HABP probe. Ofcourse, the majority of mutants and primers will not possess desirablecharacteristics, but the high-throughput screen allows those rare targetmolecules that facilitate the HA-coating process to be easilyidentified.

[0129] Biomaterials also play a pivotal role in the field of tissueengineering. Biomimetic synthetic polymers have been created to elicitspecific cellular functions and to direct cell-cell interactions both inimplants that are initially cell-free, which may serve as matrices toconduct tissue regeneration, and in implants to support celltransplantation. Biomimetic approaches have been based on polymersendowed with bioadhesive receptor-binding peptides and mono- andoligosaccharides. These materials have been patterned in two- andthree-dimensions to generate model multicellular tissue architectures,and this approach may be useful in future efforts to generate complexorganizations of multiple cell types. Natural polymers have also playedan important role in these efforts, and recombinant polymers thatcombine the beneficial aspects of natural polymers with many of thedesirable features of synthetic polymers have been designed andproduced. Biomaterials have been employed to conduct and accelerateotherwise naturally occurring phenomena, such as tissue regeneration inwound healing in the otherwise healthy subject; to induce cellularresponses that might not be normally present, such as healing in adiseased subject or the generation of a new vascular bed to receive asubsequent cell transplant; and to block natural phenomena, such as theimmune rejection of cell transplants from other species or thetransmission of growth factor signals that stimulate scar formation.

[0130] Approximately 10 years ago, the concept of bioadhesion wasintroduced into the pharmaceutical literature and has since stimulatedmuch research and development both in academia and in industry. Thefirst generation of bioadhesive drug delivery systems (BBDS) were basedon so-called mucoadhesive polymers, i.e. natural or syntheticmacromolecules, often already well accepted and used as pharmaceuticalexcipients for other purposes, which show the remarkable ability to‘stick’ to humid or wet mucosal tissue surfaces. While these noveldosage forms were mainly expected to allow for a possible prolongation,better localization or intensified contact to mucosal tissue surfaces,it had to be realized that these goals were often not so easilyaccomplished, at least not by means of such relatively straightforwardtechnology. However, although not always convincing as a “glue”, some ofthe mucoadhesive polymers were found to display other, possibly evenmore important biological activities, namely to inhibit proteolyticenzymes and/or to modulate the permeability of usually tight epithelialtissue barriers. Such features were found to be particularly useful inthe context of peptide and protein drug delivery.

[0131] The primary goal of bioadhesive controlled drug delivery is tolocalize a delivery device within the body to enhance the drugabsorption process in a site-specific manner. Bioadhesion is affected bythe synergistic action of the biological environment, the properties ofthe polymeric controlled release device, and the presence of the drugitself. The delivery site and the device design are dictated by thedrug's molecular structure and its pharmacological behavior.

[0132] One such bioadhesive known in the art is a fibrin “glue” andcompositions which include one or more types of fibrin glue incombination with a medicament have been studied. For example, in orderto test the effect on the handling properties of a two component fibringlue, the viscosity of the fibrin glue was increased with sodiumhyaluronate and the glue was applied to a microvascular anastomosis inrats. The femoral artery of each rat was anastomosed with threeconventional sutures and then sealed with the fibrin glue. Three glueswith different viscosities were tested: original Tisseel fibrin glue(Immuno AG, Vienna); Tisseel with 0.9% sodium chloride added to thefibrinogen component; and Tisseel with a high molecular weight sodiumhyaluronate (10 mg/ml, Healon, Pharmacia, Sweden) added to thefibrinogen component. The increased viscosity of the fibrin glue towhich hyaluronate had been added resulted in a significantly higherpatency rate 20 minutes after completion of the anastomosis (p<0.01),and reduced the amount of fibrin that entered the vessels. Wadstrom etal. “Fibrin glue (Tisseel) added with sodium hyaluronate inmicrovascular anastomosing.” Scand J Plast Reconstr Surg Hand SurgDecember 1993;27(4):257-61.

[0133] The typical properties of the bioadhesive fibrin system describedabove ensue from its physiological properties. Filling the woundenhances natural biological processes of healing. The tissue reaction tothe applied tissue fibrin coagulum is favorable. The treatedparenchymatous organs, liver and spleen, heal with a smooth scar. Thenumber of adhesions in the peritoneal cavity in all known treatedexperimental animals after treatment of the spleen was similar. Feweradhesions are also observed when using a bioadhesive for repairing liverinjuries in rabbits. The macroscopic appearance of the scar was similar,the scar was less visible in the liver parenchyma. The histologicalappearance was similar. The bioadhesive did not damage the tissuesurrounding the parenchyma and did not act as a foreign body. Theseresults confirm the biocompatibility of the fibrin glue as well astissue tolerance and satisfactory healing without a reaction to thebioadhesive. After healing the bioadhesive is typically replaced bynatural fibrous tissue.

[0134] Despite the effectiveness and successful use of the fibrin glueby medical practitioners in Europe, neither fibrin glue nor itsessential component fibrinogen is widely used in the United States atthe present time because of the general risks and problems of infectionfrom pooled blood products contaminated with lipid-enveloped virusessuch as HIV, associated with AIDS, and the hepatitis causing virusessuch as HBV and HCV, as well as cytomegalovirus (CMV), Epstein-Barrvirus, and the herpes simplex viruses in fibrinogen preparations. Thus,a naturally occurring or recombinantly produced bioadhesive which is notderived from pooled blood sources is actively being sought. Thebioadhesive of the present invention fulfills such a need.

[0135] For example, one embodiment of the present invention is the useof sutures or bandages with HA-chains grafted on the surface orthroughout the material in combination with the fibrinogen glue. Theimmobilized HA does not diffuse away as in current formulations, butrather remains at the wound site to enhance and stimulate healing.

[0136] Organic materials have also been postulated for use asbioadhesives. Bioadhesive lattices of water-swollen poly(acrylic acid)nano-and microparticles have been synthesized using an inverse (W/O)emulsion polymerization method. They are stabilized by a co-emulsifiersystem consisting of SpanTM 80 and TweenTM 80 dispersed in aliphatichydrocarbons. The initial polymerization medium contains emulsiondroplets and inverse micelles which solubilize a part of the monomersolution. The polymerization is then initiated by free radicals, andparticle dispersions with a narrow size distribution are obtained. Theparticle size is dependent on the type of radical initiator used. Withwater-soluble initiators, for example ammonium persulfate,microparticles are obtained in the size range of 1 to 10 micrometer,indicating that these microparticles originate from the emulsiondroplets since the droplet sizes of the W/O emulsion show similardistribution. When lipophilic radical initiators, such asazobis-isobutyronitrile, are used, almost exclusively nanoparticles aregenerated with diameters in the range of 80 to 150 nm, due to thelimited solubility of oligomeric poly(acrylic acid) chains in thelipophilic continuous phase. These poly(acrylic acid) micro- andnanoparticles yielded excellent bioadhesive properties in an in-vitroassay and may, therefore, be suitable for the encapsulation of peptidesand other hydrophilic drugs.

[0137] In the present invention, HA or chondroitin chains would be thenatural substitute for poly(acrylic-acid) based materials. HA is anegatively-charged polymer as is poly(acrylic-acid), but HA is anaturally occurring molecule in the vertebrate body and would not invokean immune response like a poly(acrylic-acid) material.

[0138] The interest in realizing ‘true’ bioadhesion continues: insteadof mucoadhesive polymers, plant or bacterial lectins, i.e. adhesionmolecules which specifically bind to sugar moieties of the epithelialcell membrane, are now widely being investigated as drug deliveryadjuvants. These second-generation bioadhesives not only provide forcellular binding, but also for subsequent endo- and transcytosis. Thismakes the novel, specifically bioadhesive molecules particularlyinteresting for the controlled delivery of DNA/RNA molecules in thecontext of antisense or gene therapy.

[0139] For the efficient delivery of peptides, proteins, and otherbiopharmaceuticals by nonparenteral routes, in particular via thegastrointestinal, or GI, tract, novel concepts are needed to overcomesignificant enzymatic and diffusional barriers. In this context,bioadhesion technologies offer some new perspectives. The original ideaof oral bioadhesive drug delivery systems was to prolong and/or tointensify the contact between controlled-release dosage forms and thestomach or gut mucosa. However, the results obtained during the pastdecade using existing pharmaceutical polymers for such purposes wererather disappointing. The encountered difficulties were mainly relatedto the physiological peculiarities of GI mucus. Nevertheless, researchin this area has also shed new light on the potential of mucoadhesivepolymers. First, one important class of mucoadhesive polymers,poly(acrylic acid), could be identified as a potent inhibitor ofproteolytic enzymes. Second, there is increasing evidence that theinteraction between various types of bio(muco)adhesive polymers andepithelial cells has direct influence on the permeability of mucosalepithelia. Rather than being just adhesives, mucoadhesive polymers maytherefore be considered as a novel class of multifunctionalmacromolecules with a number of desirable properties for their use asbiologically active drug delivery adjuvants.

[0140] In the present invention, HA or other glycosaminoglycanpolysaccharides are used. As HA is known to interact with numerousproteins (i.e. RHAMM, CD44) found throughout the healthy and diseasedbody, then naturally occurring adhesive interactions can be utilized toeffect targeting, stabilization, or other pharmacological parameters.Similarly, chondroitin interacts with a different subset of proteins(i.e. platelet factor 4, thrombin); it is likely that this polymer willyield properties distinct from HA and widen the horizon of thistechnology.

[0141] In order to overcome the problems related to GI mucus and toallow longer lasting fixation within the GI lumen, bioadhesion probablymay be better achieved using specific bioadhesive molecules. Ideally,these bind to surface structures of the epithelial cells themselvesrather than to mucus by receptor-ligand-like interactions. Suchcompounds possibly can be found in the future among plant lectins, novelsynthetic polymers, and bacterial or viral adhesion/invasion factors.Apart from the plain fixation of drug carriers within the GI lumen,direct bioadhesive contact to the apical cell membrane possibly can beused to induce active transport processes by membrane-derived vesicles(endo- and transcytosis). The nonspecific interaction between epitheliaand some mucoadhesive polymers induces a temporary loosening of thetight intercellular junctions, which is suitable for the rapidabsorption of smaller peptide drugs along the paracellular pathway. Incontrast, specific endo- and transcytosis may ultimately allow theselectively enhanced transport of very large bioactive molecules(polypeptides, polysaccharides, or polynucleotides) or drug carriersacross tight clusters of polarized epi- or endothelial cells, whereasthe formidable barrier function of such tissues against all othersolutes remains intact.

[0142] Bioadhesive systems are presently playing a major role in themedical and biological fields because of their ability to maintain adosage form at a precise body-site for a prolonged period of time overwhich the active principle is progressively released. Additional usesfor bioadhesives include: bioadhesives/mucoadhesives in drug delivery tothe gastrointestinal tract; nanoparticles as a gastroadhesive drugdelivery system; mucoadhesive buccal patches for peptide delivery;bioadhesive dosage forms for buccal/gingival administration; semisoliddosage forms as buccal bioadhesives; bioadhesive dosage forms for nasaladministration; ocular bioadhesive delivery systems; nanoparticles asbioadhesive ocular drug delivery systems; and bioadhesive dosage formsfor vaginal and intrauterine applications.

[0143] The bioadhesive may also contain liposomes. Liposomes areunilamellar or multilamellar lipid vesicles which entrap a significantfraction of aqueous solution. The vesicular microreservoirs of liposomescan contain a variety of water-soluble materials, which are thussuspended within the emulsion. The preparation of liposomes and thevariety of uses of liposomes in biological systems has been disclosed inU.S. Pat. Nos. 4,708,861, 4,224,179, and 4,235,871. Liposomes aregenerally formed by mixing long chain carboxylic acids, amines, andcholesterol, as well as phospholipids, in aqueous buffers. The organiccomponents spontaneously form multilamellar bilayer structures calledliposomes. Depending on their composition and storage conditions,liposomes exhibit varying stabilities. Liposomes serve as models of cellmembranes and also are Used as drug delivery systems.

[0144] Most attempts to use liposomes as drug delivery vehicles haveenvisioned liposomes as entities which circulate in blood, to be takenup by certain cells or tissues in which their degradation would slowlyrelease their internal aqueous drug-containing contents. In an effort toaid in their up-take by a given target tissue, some liposomes have been“tailored” by binding specific antibodies or antigens to the outersurface. Liposomes have also been devised as controlled release systemsfor the delivery of their contents in vivo. Compositions in whichliposomes containing biologically active agents are maintained andimmobilized in polymer matrices, such as methylcellulose, collagen andagarose, for sustained release of the liposome contents, are describedin U.S. Pat. No. 4,708,861 to Popescu et al.

[0145] In this manner, the present invention contemplates a bioadhesivecomprising HA produced from PmHAS. The present invention alsocontemplates a composition containing a bioadhesive comprising HAproduced from PmHAS and an effective amount of a medicament, wherein themedicament can be entrapped or grafted directly within the HAbioadhesive or be suspended within a liposome which is entrapped orgrafted within the HA bioadhesive. These compositions are especiallysuited to the controlled release of medicaments.

[0146] Such compositions are useful on the tissues, skin, and mucusmembranes (mucosa) of an animal body, such as that of a human, to whichthe compositions adhere. The compositions so adhered to the mucosa,skin, or other tissue slowly release the treating agent to the contactedbody area for relatively long periods of time, and cause the treatingagent to be sorbed (absorbed or adsorbed) at least at the vicinity ofthe contacted body area. Such time periods are longer than the time ofrelease for a similar composition that does not include the HAbioadhesive.

[0147] The treating agents useful herein are selected generally from theclasses of medicinal agents and cosmetic agents. Substantially any agentof these two classes of materials that is a solid at ambienttemperatures may be used in a composition or method of the presentinvention. Treating agents that are liquid at ambient temperatures, e.g.nitroglycerine, can be used in a composition of this invention, but arenot preferred because of the difficulties presented in theirformulation. The treating agent may be used singly or as a mixture oftwo or more such agents.

[0148] One or more adjuvants may also be included with a treating agent,and when so used, an adjuvant is included in the meaning of the phrase“treating agent” or “medicament.” Exemplary of useful adjuvants arechelating agents such as EDTA that bind calcium ions and assist inpassage of medicinal agents through the mucosa and into the bloodstream. Another illustrative group of adjuvants are the quaternarynitrogen-containing compounds such as benzalkonium chloride that alsoassist medicinal agents in passing through the mucosa and into the bloodstream.

[0149] The treating agent is present in the compositions of thisinvention in an amount that is sufficient to prevent, cure and/or treata condition for a desired period of time for which the composition ofthis invention is to be administered, and such an amount is referredherein as “an effective amount.” As is well known, particularly in themedicinal arts, effective amounts of medicinal agents vary with theparticular agent involved, the condition being treated and the rate atwhich the composition containing the medicinal agent is eliminated fromthe body, as well as varying with the animal in which it is being used,and the body weight of that animal. Consequently, effective amounts oftreating agents may not be defined for each agent. Thus, an effectiveamount is that amount which in a composition of this invention providesa sufficient amount of the treating agent to provide the requisiteactivity of treating agent in or on the body of the, treated animal forthe desired period of time, and is typically less than that amountusually used.

[0150] Inasmuch as amounts of particular treating agents in the bloodstream that are suitable for treating particular conditions aregenerally known, as are suitable amounts of treating agents used incosmetics, it is a relatively easy laboratory task to formulate a seriesof controlled release compositions of this invention containing a rangeof such treating agent for a particular composition of this invention.

[0151] The second principle ingredient of this embodiment of the presentinvention is a bioadhesive comprising an amount of hyaluronic acid (HA)from PmHAS or chondroitin from PmCS. Such a glycosaminoglycanbioadhesive made from a HA or chondroitin chain directly polymerizedonto a molecule with the desired pharmacological property or a HA orchondroitin chain polymerized onto a matrix or liposome which in turncontains or binds the medicament.

[0152] Although the foregoing invention has been described in somedetail by way of illustration and example for purposes of clarity ofunderstanding, it will be obvious to those skilled in the art thatcertain changes and modifications may be practiced without departingfrom the spirit and scope thereof, as described in this specificationand as defined in the appended claims below.

What I claim is:
 1. A method for transferring sugars to a primermolecule or an acceptor molecule utilizing an enzymatic process.
 2. Themethod according to claim 1, wherein the primer molecule or the acceptormolecule is a short polysaccharide.
 3. The method according to claim 2,wherein the polysaccharide is an oligosaccharide.
 4. The methodaccording to claim 1, wherein the primer molecule or the acceptormolecule is a long polysaccharide.
 5. The method according to claim 1,wherein the primer molecule or the acceptor molecule is a solidsubstrate.
 6. The method according to claim 5, wherein the solidsubstrate is selected from the group consisting of a semiconductor, ametal, a natural or man-made polymer, and combinations thereof.
 7. Themethod according to claim 1, wherein the enzymatic process utilizes apolysaccharide synthase.
 8. The method according to claim 1, wherein theenzymatic process utilizes a glycosaminoglycan synthase.
 9. The methodaccording to claim 1, wherein the enzymatic process utilizes ahyaluronic acid synthase.
 10. The method according to claim 9, whereinthe hyaluronic acid synthase is selected from the group consisting ofPmHAS-D, mutant variations of PmHAS, chemically modified versions ofPmHAS, and combinations thereof.
 11. The method according to claim 10,wherein the amino acid sequence of the PmHAS-D is in accordance with SEQID NO:1.
 12. The method according to claim 10, wherein the nucleotidesequence of the PmHAS-D is in accordance with SEQ ID NO:2.
 13. Themethod of claim 1, wherein the enzymatic process utilizes a chondroitinsynthase.
 14. The method of claim 13, wherein the chondroitin synthaseis selected from the group consisting of PmCS, mutant variations ofPmCS, chemically modified versions of PmCS, and combinations thereof.15. The method of claim 14, wherein the amino acid sequence of the PmCSis in accordance with SEQ ID NO:
 3. 16. The method of claim 14, whereinthe nucleotide sequence of the PmCS is in accordance with SEQ ID NO: 4.17. The method of claim 1, wherein the sugars are selected from thegroup consisting of a natural or man-made monosaccharide, a GlcNAc, aGlcA, a GalNAc, a GalA, and combinations thereof.
 18. The method ofclaim 1, wherein the enzymatic process utilizes a polysaccharidesynthase which has been altered by at least one condition selected fromthe group consisting of natural variations or variants, chemicalmodification, genetic modification, altered reaction conditions, andcombinations thereof.
 19. A purified nucleic acid segment having acoding region encoding enzymatically active PmHAS-D.
 20. The purifiednucleic acid segment of claim 19, wherein the purified nucleic acidsegment comprises a nucleotide sequence in accordance with SEQ ID NO:2.21. A purified nucleic acid segment having a coding region encodingenzymatically active PmHAS-D, wherein the purified nucleic acid segmentis capable of. hybridizing to the nucleotide sequence of SEQ ID NO:2.22. A recombinant vector selected from the group consisting of aplasmid, a cosmid, a phage a virus vector, or a chromosomally integratedexpression cassette and wherein the recombinant vector further comprisesa purified nucleic acid segment having a coding region encodingenzymatically active PmHAS-D.
 23. The recombinant vector of claim 22wherein the purified nucleic acid segment comprises a nucleotidesequence in accordance with SEQ ID NO:2.
 24. The recombinant vector ofclaim 22, wherein the recombinant vector is a plasmid.
 25. Therecombinant vector of claim 24, wherein the plasmid further comprises anexpression vector.
 26. The recombinant vector of claim 25, wherein theexpression vector comprises a promoter operatively linked to theenzymatically active PmHAS-D coding region.
 27. A recombinant host cell,Wherein the recombinant host cell is a prokaryotic cell transformed witha recombinant vector comprising a purified nucleic acid segment having acoding region encoding enzymatically active PmHAS-D.
 28. A method forproducing a polysaccharide polymer on a substrate, comprising the stepsof: providing a substrate having an immobilized polysaccharide primerthereon to provide a primed substrate; combining the primed substratewith a PmHAS-D enzyme within a reaction medium, wherein the reactionmedium contains at least one sugar precursor selected from the groupconsisting of UDP-GlcA and UDP-GlcNAc; and reacting the PmHAS-D enzymewith the primed substrate to produce a substrate having a polysaccharidepolymer coated thereon.
 29. The method of claim 28, wherein thepolysaccharide polymer is hyaluronic acid.
 30. The method of claim 28,wherein the polysaccharide polymer is chondroitin.
 31. A substratecoated with a polysaccharide polymer manufactured according to theprocess of claim
 28. 32. The substrate of claim 31, wherein thepolysaccharide polymer is hyaluronic acid.
 33. The substrate of claim31, wherein the polysaccharide polymer is chondroitin.
 34. A method forproducing a polysaccharide bioadhesive sealant, comprising the steps of:combining an enzyme selected from the group consisting of PmHAS-D andPmCS with a reaction medium, wherein the reaction medium contains atleast one sugar precursor selected from the group consisting ofUDP-GlcA, UDP-GlcNAc or UDP-GalNAc; and allowing the enzyme to reactwith the reaction medium, wherein the enzyme produces a polysaccharidepolymer capable of use as a bioadhesive sealant by successivelycombining the at least one sugar precursor into a polymer chain.
 35. Apolysaccharide bioadhesive sealant manufactured according to the processof claim
 34. 36. A method for producing a polysaccharide biomaterialcontaining a medicament delivery assembly, comprising the steps of:preparing a polysaccharide polymer from an enzyme selected from thegroup consisting of PmHAS or PmCS, wherein the polysaccharide polymer iscapable of acting as a bioadhesive; providing at least one medicamentdelivery assembly containing one or more medicaments entrapped thereinand deliverable within a wound site or a surgical site; and mixing theprepared polysaccharide bioadhesive with the at least one medicamentdelivery assembly, wherein the prepared polysaccharide bioadhesiveentraps the at least one medicament delivery. assembly to produce apolysaccharide biomaterial containing a medicament delivery system. 37.A polysaccharide biomaterial containing a medicament delivery assemblymanufactured according to the process of claim
 36. 38. A method forproducing a polysaccharide biomaterial containing a medicament deliverysystem for administration at a wound, ulcer, injury or surgical site,comprising the steps of: preparing a polysaccharide polymer from aenzyme selected from the group consisting of PmHAS or PmCS, wherein theprepared polysaccharide polymer is capable of acting as a bioadhesive;providing at least one medicament delivery assembly containing one ormore medicaments entrapped therein; and mixing the preparedpolysaccharide bioadhesive with the at least one medicament deliveryassembly, wherein the prepared polysaccharide bioadhesive entraps the atleast one medicament delivery assembly to provides a polysaccharidebiomaterial containing a medicament delivery assembly.
 39. Apolysaccharide biomaterial containing a medicament delivery systemmanufactured according to the process of claim
 38. 40. A method forinducing coagulation of a medicament delivery assembly-containingpolysaccharide biomaterial composition at a site of injury, wound, orsurgery in a mammal, thereby resulting in sealing and healing of theinjury, wound or surgical site, comprising the steps of: preparing amedicament delivery assembly-containing polysaccharide bioadhesivecomposition from an enzyme selected from the group consisting of PmHASor PmCS; and administering the medicament delivery assembly-containingpolysaccharide bioadhesive composition to a mammal, wherein thecoagulation of the medicament delivery assembly-containingpolysaccharide bioadhesive composition forms a medium for entrapping themedicament delivery assembly at the site and embeds the medicamentdelivery assembly in the polysaccharide biomaterial composition.
 41. Amethod for hemostatically sealing and healing a wound or surgicalincision and delivering to the wound or incision medicaments, comprisingthe steps of: preparing a polysaccharide biomaterial composition havinga medicament containing delivery assembly, wherein the polysaccharidebioadhesive composition is prepared from an enzyme selected from thegroup consisting of PmHAS and PmCS; and administering the composition tothe surface of a wound or incision, wherein coagulation of thecomposition forms a medium for entrapping the medicament deliveryassembly at the site, embeds the medicament delivery assembly in thecomposition, and allows for a sustained release of the medicamentdelivery assembly contents to the wound or incision.
 42. A biomaterialcomposition, comprising: an effective amount of a polysaccharide polymerproduced from an enzyme selected from the group consisting of PmHAS-Dand PmCS; and at least one medicament delivery assembly containing atleast one medicament, wherein the at least one medicament deliveryassembly is embedded in the polysaccharide polymer so as to localize theat least one medicament within the polysaccharide polymer bioadhesivecomposition.